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WO2013013068A2 - Modes de réalisation d'une sonde et procédé de ciblage d'acides nucléiques - Google Patents

Modes de réalisation d'une sonde et procédé de ciblage d'acides nucléiques Download PDF

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Publication number
WO2013013068A2
WO2013013068A2 PCT/US2012/047442 US2012047442W WO2013013068A2 WO 2013013068 A2 WO2013013068 A2 WO 2013013068A2 US 2012047442 W US2012047442 W US 2012047442W WO 2013013068 A2 WO2013013068 A2 WO 2013013068A2
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WIPO (PCT)
Prior art keywords
probe
human
monomer
target
nucleic acid
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Application number
PCT/US2012/047442
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English (en)
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WO2013013068A3 (fr
WO2013013068A4 (fr
Inventor
Patrick J. Hrdlicka
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University Of Idaho
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Publication date
Application filed by University Of Idaho filed Critical University Of Idaho
Priority to CA2842103A priority Critical patent/CA2842103A1/fr
Priority to US14/233,375 priority patent/US9885082B2/en
Priority to RU2014106024/04A priority patent/RU2014106024A/ru
Priority to NZ621347A priority patent/NZ621347B2/en
Priority to AU2012283994A priority patent/AU2012283994A1/en
Priority to MX2014000797A priority patent/MX2014000797A/es
Priority to BR112014001075A priority patent/BR112014001075A2/pt
Priority to EP12814839.2A priority patent/EP2734525A4/fr
Priority to CN201280043607.7A priority patent/CN103958519A/zh
Publication of WO2013013068A2 publication Critical patent/WO2013013068A2/fr
Publication of WO2013013068A3 publication Critical patent/WO2013013068A3/fr
Publication of WO2013013068A4 publication Critical patent/WO2013013068A4/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6879—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for sex determination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
    • Y10T436/143333—Saccharide [e.g., DNA, etc.]

Definitions

  • Disclosed herein are embodiments of a probe capable of targeting nucleic acids and particular sequences thereof. Also disclosed are methods for using the disclosed probe to target nucleic acids and sequences thereof.
  • dsDNA double stranded DNA
  • PNAs peptide nucleic acids
  • pcDNA pseudo-complementary DNA
  • pcPNA pseudo- complementary PNA
  • Particular disclosed embodiments concern a probe, comprising: a pair of monomers comprising a first monomer having a formula
  • each Y independently is selected from carbon, oxygen, sulfur, a triazole, and NR b , wherein R b is selected from hydrogen, aliphatic, aryl, heteroaliphatic, and heteroaryl; V is selected from carbon, oxygen, sulfur, and NR b ; n ranges from 0 to 4; R 1 and R 2 are selected from hydrogen, aliphatic, aryl, and a heteroatom-containing moiety; R 3 is a heteroatom-containing functional group; R 4 is selected from any natural or non-natural nucleobase; R 5 is selected from any aromatic moiety suitable for intercalating within a nucleic acid; "optional linker" is selected from alkyl, amide, carbamate, carbonate, urea, and combinations thereof; a second monomer having a formula
  • At least one nucleotide selected from a natural nucleotide, a non-natural nucleotide, and combinations thereof, wherein the at least one nucleotide typically is coupled to the first and/or second monomer at R 2 and/or R 3 by a phosphate group.
  • the heteroatom-containing functional group is selected from ether (R a OR b ), hydroxyl (ROH), silyl ether (R a R b R c SiOR d ), phosphine (PR a R b R c ), thiol (R SH), thioether/sulfide (R a SR b ), disulfide (R a SSR b ), isothiocyanate (R NCS), isocyanate (R NCO), amine (NH 2 , NHR , NR a R b ), amide (R NR b C(0)R c ), ester (R a OC(0)R b ), halogen (I, Br, CI, F), carbonate (R a OC(0)OR b ), carboxyl (R a C(0)OH), carboxylate (R a COO ), ester (R a C(0)OR b ), ketone
  • R a C(0)R b phosphate (R a OP(0)OH 2 ), phosphoryl (R a P(0)(OH) 2 ), sulfinyl (R a S(0)R b ), sulfonyl (R a S0 2 R b ), carbonothioyl (R a C(S)R b or R a C(S)H), sulfino (R a S(0)OH), sulfo (R a S0 3 H), amide (R C(0)NR b R c ), azide (N 3 ), nitrile (R CN), isonitrile (R a N + C ⁇ ), and nitro (R a N0 2 );
  • R a represents the remaining monomer structure, which is attached to the abovementioned functional groups at the position indicated; and
  • R b , R c , and R d independently are hydrogen, aliphatic, aryl, heteroaliphatic, heteroaryl,
  • R 2 and R 3 independently are selected from a heteroatom functional group comprising phosphorous, sulfur, nitrogen, oxygen, selenium, and/or a metal, more typically R 2 and R 3 independently are selected from a phosphate group of the natural nucleotide, non-natural nucleotide, synthetic nucleotides, or combinations thereof.
  • R 4 is selected from adenine, guanine, cytosine, uracil, thymine, or any derivative thereof and the intercalator is an aromatic hydrocarbon or an aromatic heterocycle.
  • the intercalator is a hydrocarbon selected from pyrene, coronene, perylene, anthracene, naphthalene and functionalized derivatives thereof; or may be an aromatic heterocycle selected from a porphyrin, nucleobase, metal chelator, azapyrene, thiazole orange, indole, pyrrole, and derivatives thereof.
  • Certain disclosed embodiments concern a probe having at least one monomer, and typically two or more monomers.
  • the monomers are generally incorporated within oligonucleotide strands, and can be located anywhere in the oligonucleotide strand, including the first position, the last position, and anywhere in between, and preferably are arranged in a final structure in an interstrand zipper arrangement, such as a +1 zipper arrangement, as dicussed in more detail below.
  • Certain disclosed embodiments concern probes wherein either the first monomer or the second monomer has a formula
  • either the first monomer or the second monomer has a formula
  • B is selected from uracil, guanine, cytosine, adenine, thymine, 2-thiouracil, 2,6- diaminopurine, inosine, 3-pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one or derivatives thereof.
  • B is selected from uracil, guanine, cytosine, adenine, thymine, 2-thiouracil, 2,6- diaminopurine, inosine, 3-pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one or derivatives thereof
  • R e is H, DMTr, or phosphate, such as provided by a phosphodiester bond in an oligonucleotide and R f is H, (N(i-Pr) 2 )P(OCH 2 CH 2 CN), or phosphate, such as provided by a phosphodiester bond in an oligonucleotide
  • Nap refers to napthyl, such as 2-napthyl, Cor to coronenyl, such as coronen- 1 -yl, Py to pyrenyl, such as pyren-l-yl, pyren-2-yl and pyren-4-yl and Pery to peryleneyl,
  • the at least one natural nucleotide may be selected from adenine, guanine, cytosine, uracil, thymine and derivatives thereof
  • the at least one non- natural nucleobase may be selected from C5-functionalized pyrimidines, C6-functionalized pyrimidines, C7-functionalized 7-deazapurines, C8-functionalized purines, 2-thiouracil, 2,6- diaminopurine, inosine, 3-pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one or derivatives thereof.
  • the probe comprises at least one natural nucleotide, unnatural nucleotide, and combinations thereof, which are selected to substantially match at least one nucleotide of a corresponding nucleic acid sequence.
  • B 1; B 2 and B m . s may be any natural or non-natural nucleotide, wherein m-s ranges from zero to about 28;/, g, h, i and j range from 0 to 10, more likely 0 to 5, and typically 0 to 3;
  • X is the first monomer;
  • A is the complement Watson-Crick base pairing nucleotide of P;
  • C is any natural or non- natural nucleotide capable of Watson-Crick base pairing with any one of B B 2 and B m . s ;
  • P is the second monomer, and D is the complement Watson-Crick base pairing nucleotide of X.
  • the probe is selected to recognize a predetermined sequence of a nucleic acid target, which may be single-stranded or double-stranded, more commonly double-stranded.
  • the probe may additionally comprise one or more monomers at any given position that do not participate in base pairing, such as the following structures, wherein R e is H, DMTr, or phosphate, such as provided by a phosphodiester bond in an oligonucleotide and R f is H, (N(i-Pr) 2 )P(OCH 2 CH 2 CN), or phosphate, such as provided by a phosphodiester bond in an oligonucleotide, and R s and R h are, by way of example and without limitation, independently selected from hydrogen, aliphatic, particularly alkyl, such as CI -CIO alkyl, cyclic, heterocyclic, aromatic, heteroaromatic, amides, and carbamates .
  • the probe may have a thermal melting temperature which is comparable (slightly lower, similar or slightly higher) to that of a corresponding (i.e., isosequential) unmodified nucleic acid duplex, which typically does not comprise a monomer having the formulas disclosed herein.
  • the probe may comprise a pair of monomers wherein the first monomer and the second monomer are arranged in a +n or -n interstrand zipper orientation, wherein n ranges from 0 to about 10, more typically from 0 to about 2, more typically the first monomer and the second monomer are arranged in a +n orientation, wherein n is 1.
  • the probe may also comprise one or more additional pairs of monomers; a signal generating moiety capable of being detected, selected from a fluorophore, a member of a specific binding pair (e.g.
  • the secondary entity facilitates cell- uptake and/or cellular compartmentalization and includes peptides [NLS, CPP, KDEL], and small molecules with nuclear affinity (e.g.
  • the probe may be used in solution, on a solid surface (e.g. multi-well plates; noble metal surfaces, such as electrodes), or in combination with a colloidal material and/or nanomaterials (e.g. gold nanoparticles, quantum dots).
  • a solid surface e.g. multi-well plates; noble metal surfaces, such as electrodes
  • nanomaterials e.g. gold nanoparticles, quantum dots
  • Certain disclosed embodiments concern a method for detecting a target, comprising:
  • the probe is selected to substantially match a region of the target nucleic acid, particularly double stranded nucleic acid target regions, which may or may not comprise one or more polypurinestretches; more typically the nucleic acid is a mixed sequence of nucleotides, structured nucleic acid, particularly double-stranded nucleic acid sequences (dsDNA), even more particularly double stranded DNA, such as by way of example a mixed-sequence, hairpin DNA targets, PCR amplicons, genomic DNA, etc. which are isosequential with the probe.
  • exposing the target to the probe comprises incubating the probe with the nucleic acid target.
  • the nucleic acid target is incubated with an excess, such as about a 5-fold excess of the probe up to at least about a 5,000-fold excess of the probe, more typically up to about a 500-fold excess of the probe, and even more typically about a 5-fold excess of the probe to about a 200-fold excess of the probe.
  • the probe and probe-target (recognition) complex is detected by fluorescence spectroscopy, electrophoresis, absorption spectroscopy, fluorescence microscopy, flow cytometer, and combinations thereof.
  • the target sequences for disclosed probe embodiments can vary.
  • the method is particularly useful for gender determination in mammals.
  • the method can be used for gender determination of ungulates and ruminates, particularly bovines, equines or porcines.
  • the target is isosequential (relative to a probe) to double stranded DNA target regions, including stems of molecular beacons, target regions embedded within PCR amplicons, target regions embedded within circular or linearized plasmids, target regions embedded within genomic DNA, and target regions embedded within microorganisms.
  • the target also can be selected from a nucleic acid sequence associated with a proliferative disorder, such as B cell and T cell leukemias, lymphomas, breast cancer, colon cancer, and neurological cancers.
  • a proliferative disorder such as B cell and T cell leukemias, lymphomas, breast cancer, colon cancer, and neurological cancers.
  • the target is selected from a virus or other microorganism and the probe is used to detect and/or identify the microorganism.
  • kits typically comprise a probe comprising at least one disclosed monomer, and may comprise at least one bulge monomer.
  • kits also may include a sequence selected from SEQ ID NOs. 1-254. Certain kit embodiments are particularly useful for gender determination in mammals.
  • FIG. 1 is a schematic diagram that illustrates a particular embodiment of a method for detecting a target using the disclosed probe.
  • FIG. 2 is an image of an embodiment of the disclosed probe.
  • FIG. 3A is a schematic drawing illustrating the concept of using bulge monomers.
  • FIG. 3B is a schematic drawing illustrating probes with two +1 interstrand arrangements of disclosed monmers (green) and one to four non-pairing bulged monomers (red loop), such as 402-4, 402-N and 402-9.
  • FIG. 4 is an image of a thermal denaturation curve between an exemplary embodiment of disclosed probes and a nucleic acid target illustrating characteristics of duplexes between one of the (two) probe strands and one of the (two) target strands.
  • FIG. 5 is an image of a thermal denaturation curve between an exemplary embodiments of disclosed probes and a nucleic acid target illustrating characteristics of duplexes between one of the (two) probe strands and one of the (two) target strands.
  • FIG. 6 is an image of a thermal denaturation curve between an exemplary embodiment of disclosed probes and a nucleic acid target illustrating characteristics of duplexes between one of the (two) probe strands and one of the (two) target strands.
  • FIG. 7 are absorption spectra obtained from an exemplary embodiment and duplexes formed with a nucleic acid target.
  • FIG. 8 are absorption spectra obtained from an exemplary probe embodiment and the duplexes formed with a nucleic acid targets.
  • FIG. 9 are fluorescence emission spectra of an exemplary probe embodiment in the presence or absence of a nucleic acid target.
  • FIG. 10 are fluorescence emission spectra of an exemplary probe embodiment in the presence or absence of a nucleic acid target.
  • FIG. 11 are fluorescence emission spectra of an exemplary probe embodiment in the presence or absence of a nucleic acid target.
  • FIG. 12 are fluorescence emission spectra of an exemplary probe embodiment in the presence or absence of a nucleic acid target.
  • FIG. 13 are fluorescence emission spectra of an exemplary probe embodiment in the presence or absence of a nucleic acid target.
  • FIG. 14 are fluorescence emission spectra of an exemplary probe embodiment in the presence or absence of a nucleic acid target.
  • FIG. 15 are fluorescence emission spectra of an exemplary probe embodiment in the presence or absence of a nucleic acid target.
  • FIG. 16 are fluorescence emission spectra of an exemplary probe embodiment in the presence or absence of a nucleic acid target.
  • FIG. 17 are fluorescence emission spectra of an exemplary probe embodiment in the presence or absence of a nucleic acid target.
  • FIG. 18 is a schematic drawing of a target used in an exemplary method of using a disclosed probe.
  • FIG. 19 is a schematic drawing of a target and exemplary embodiments of the disclosed probe used in an exemplary method of using the probe.
  • FIG. 20 is an image of a gel obtained using gel electrophoresis analysis of different exemplary embodiments of the probe and their ability to bind to a particular target.
  • FIG. 21 is schematic drawing of a target used to analyze the ability of the disclosed probe to suppress gene expression.
  • FIG. 22 is schematic drawing of exemplary embodiments of disclosed probes and the results obtained using such probes to suppress gene expression in a particular target.
  • FIG. 23 is schematic drawing of the results obtained from a dose-dependent study using exemplary embodiments of the disclosed probe and a particular target.
  • FIG. 24 is schematic drawing of an exemplary embodiment wherein the probe is used to target a mixed-sequence, structured nucleic acid target.
  • FIG. 25 is an image of a gel obtained from gel electrophoresis analysis of an exemplary embodiment of the disclosed probe illustrating its ability to complex with a mixed-sequence, structured nucleic acid target.
  • FIG. 26 is an image of the results obtained using a variety of exemplary embodiments of the disclosed probes as well as embodiments used as controls (e.g. single-stranded probe precursor and a control with no probe) to target and form a complex with a mixed-sequence, structured nucleic acid target.
  • controls e.g. single-stranded probe precursor and a control with no probe
  • FIG. 27 are absorption spectra obtained using exemplary embodiments of a disclosed probe comprising a triazole moiety.
  • FIG. 28 are steady-state fluorescence emission spectra obtained using exemplary embodiments of a disclosed probe comprising a triazole moiety.
  • FIG. 29 is schematic drawing illustrating a putative mechanism of the disclosed probe wherein universal hybridization occurs.
  • FIG. 30 illustrates recognition of structured dsDNA targets by probes using electrophoretic mobility shift assays: (a) schematic llustration of recognition process; (b) structures of dsDNA- targets with isosequential (SL1) or non-isosequential (SL2 and SL3) stem regions (arrows denote deviation points); (c), (d) and (e) recognition of SL1 using escalating excess of 126W2:126W5, 120Q2:120Q5 or D1:D2, respectively; (f) incubation of SL1 with 100-fold excess of single-stranded 126W2, 126W5, 120Q2 or 120Q5; (g) and (h) incubation of SL1-SL3 with 100-fold excess of 126W2:126W5 or 120Q2:120Q5, respectively. Probe-target incubation: 3 hours at 20 °C; 15% non- denaturing PAGE; DIG: digoxigenin.
  • FIG. 31 are dose-response curves for recognition of structured dsDNA SL1 by the following probes (top to bottom): 126W2:126W5, 126X2:126X5, 126Y2:126Y5, 120Q2:120Q5,
  • FIG. 32 illustrates the results of a control experiment. Incubation of SL1 with 100-fold excess of single-stranded 120'W6 (M6), 120'W8 (M8), 120Y2 (P2) or 120Y5 (P5). For experimental conditions and sequence of SL1, see FIG. 30.
  • FIG. 33 illustrates the results of a control experiment. Incubation of SL1-SL3 with 100- fold excess of a) 126X2:126X5, b) 126Y2:126Y5 or c) 124X6:124X8. For experimental conditions and sequence of SL1-SL3, see FIG. 30.
  • FIG. 34 illustrates targeting of structured dsDNA HPl using different probes modified with monomer 120Y (200-fold molar excess) as monitored by the gel mobility shift assay (electrophoretic mobility shift assay; for concept see FIG 30).
  • HPl (5 ' -GGTATATATAGGC-(T 10)- GCCTATATATACC (34.4 nM) incubated at room temperature.
  • HPl alone (lane 1); HPl incubated with 200-excess of 120Y-P1 (lane 2), 120Y-P2 (lane 3), 120Y-P3 (lane 4), 120Y-P4 (lane 5), 120Y- P5 (lane 6), 120Y-P6 (lane 7) or 120Y-P7 (lane 8).
  • FIG. 35 illustrates targeting of structured dsDNA HPl using various concentrations of a selected probe (120Y-P4).
  • FIG. 36 illustrates the results of a control experiment demonstrating the recognition specificity of the disclosed probes.
  • FIG. 37 provides proof for a proposed recognition mechanism, demonstrating the disclosed mode of nucleic acid targeting using disclosed probes.
  • Lane 1 DIG-labeled HPl only;
  • lane 2 DIG- labeled 'upper' probe strand only (5'- GG(120Y)A(120Y)A TAT AGG C-DIG);
  • lane 3 DIG-labeled 'lower' probe strand only (3'- DIG-CCA (120Y)A(120Y) ATA TCC G);
  • lane 4 probe with DIG- labeled upper strand (5 ' -GG(120Y) A(120Y) ATATAGGC-DIG + 3'-
  • lane 5 probe with DIG-labeled lower strand (5 ' -GG(120Y) A(120Y) ATATAGGC + 3'-DIG- CCA(120YW120Y)ATATCCG) incubated with unlabeled structured target HPl; lane 6: unlabeled probe (5'-GG(120Y)A(120Y)ATATAGGC + 3'-CCA(120Y)A(120Y)ATATCCG) incubated with labeled structured target HPl .
  • FIG. 38 provides information that both strands of a double-stranded probe facilitate recognition of dsDNA target regions.
  • Lane 1 only HPl; lane 2: HPl + upper strand of 120Y-P4 (i.e., 5'-GG(120Y)A(120Y)ATATAGGC); lane 3: HPl + lower strand of 120Y-P4 (i.e., 3'- CCA(120Y)A(120Y)ATATCCG); lane 4: HPl + 120Y-P4.
  • Probes are used in 200-fold molar excess relative to structured target HPl. Incubated in Hepes buffer for 15 hours at room temperature.
  • FIG. 39 is a gel illustrating the results of incubation of HPl with increasing concentrations of an isosequential and unmodified dsDNA probe.
  • 5x-500x refers to molar fold excess of dsDNA with respect to HPl
  • FIG. 40 is a graph of linker chemistry versus invastion% for symmetric bulges illustrating the results of targeting structured dsDNA (stem-loop) target using probe with one or more bulges, where Dig-labeled structured dsDNA target was incubated with 200-fold excess of probe in Hepes buffer for 15 hours followed by electrophoresis, imaging, and quantification.
  • FIG. 41 is a graph of linker chemistry versus invastion% for an up stranded bulge illustrating the results of targeting structured dsDNA (stem-loop) target using probe with one or more bulges where Dig-labeled structured dsDNA target was incubated with 200-fold excess of probe in Hepes buffer for 15 hours followed by electrophoresis, imaging, and quantification.
  • FIG. 42 is a graph of linker chemistry versus invastion% for a down stranded bulge illustrating the results of targeting structured dsDNA (stem-loop) target using probe with one or more bulges where Dig-labeled structured dsDNA target was incubated with 200-fold excess of probe in Hepes buffer for 15 hours followed by electrophoresis, imaging, and quantification.
  • FIG. 43 is an illustration on the use of double-stranded probes with +1 interstrand zipper arrangements that are additionally modified with a fluorophore-quencher pair. Binding to the nucleic acid target results in generation of an optical signal, more commonly a fluorescent signal.
  • nucleic acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
  • SEQ ID NOs: 1-4, 11 and 240-245 are nucleotide sequences of structured dsDNA targets with isosequential or non-isosequential stem regions.
  • SEQ ID Nos: 5-10, 12-239, 246-251 and 254 are nucleotide sequences of oligonucleotide probes and target regions.
  • SEQ ID Nos: 252 and 253 are nucleotide sequences of double-stranded probes containing a non-pairing bulge.
  • a wavy line ") indicates a bond disconnection.
  • a dashed line (“ ") illustrates that a bond may be formed at a particular position.
  • nucleotide sizes or amino acid sizes and all molecular weight or molecular mass values, given for nucleic acids or polypeptides or other compounds are approximate, and are provided for description.
  • Aliphatic Any open or closed chain molecule, excluding aromatic compounds, containing only carbon and hydrogen atoms which are joined by single bonds (alkanes), double bonds (alkenes), or triple bonds (alkynes). This term encompasses substituted aliphatic compounds, saturated aliphatic compounds, and unsaturated aliphatic compounds.
  • Analog, Derivative or Mimetic An analog is a molecule that differs in chemical structure from a parent compound, for example a homolog (differing by an increment in the chemical structure, such as a difference in the length of an alkyl chain), a molecular fragment, a structure that differs by one or more functional groups, a change in ionization. Structural analogs are often found using quantitative structure activity relationships (QSAR), with techniques such as those disclosed in Remington (The Science and Practice of Pharmacology, 19th Edition (1995), chapter 28).
  • a derivative is a biologically active molecule derived from the base structure.
  • a mimetic is a molecule that mimics the activity of another molecule, such as a biologically active molecule.
  • Biologically active molecules can include chemical structures that mimic the biological activities of a compound.
  • Aromatic A term describing conjugated rings having unsaturated bonds, lone pairs, or empty orbitals, which exhibit a stabilization stronger than would be expected by the stabilization of conjugation alone. It can also be considered a manifestation of cyclic delocalization and of resonance.
  • Aryl A substantially hydrocarbon-based aromatic compound, or a radical thereof (e.g.
  • CeH 5 as a substituent bonded to another group, particularly other organic groups, having a ring structure as exemplified by benzene, naphthalene, phenanthrene, anthracene, etc. This term also encompasses substituted aryl compounds.
  • Aryl alkyl A compound, or a radical thereof (C 7 H 7 for toluene) as a substituent bonded to another group, particularly other organic groups, containing both aliphatic and aromatic structures.
  • Complementary The natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. Complementarity may exist when only some of the nucleic acids bind, or when total complementarity exists between the nucleic acids.
  • Conjugating, joining, bonding or linking Joining one molecule to another molecule to make a larger molecule. For example, making two polypeptides into one contiguous polypeptide molecule, or covalently attaching a hapten or other molecule to a polypeptide, such as an scFv antibody.
  • the terms include reference to joining a ligand, such as an antibody moiety, to an effector molecule.
  • the linkage can be either by chemical or recombinant means.
  • “Chemical means” refers to a reaction between the antibody moiety and the effector molecule such that there is a covalent bond formed between the two molecules to form one molecule.
  • Coupled means joined together, either directly or indirectly.
  • a first atom or molecule can be directly coupled or indirectly coupled to a second atom or molecule.
  • a secondary antibody provides an example of indirect coupling. Coupling can occur via covalent, non- covalent, and ionic bond formation.
  • a derivative In chemistry, a derivative is a compound that is derived from a similar compound or a compound that can be imagined to arise from another compound, for example, if one atom is replaced with another atom or group of atoms. The latter definition is common in organic chemistry. In biochemistry, the word is used for compounds that at least theoretically can be formed from the precursor compound.
  • Deviation from additivity The DA value for a probe ONX:ONY is defined as: a :ONY ⁇ &T m (ONX:ONY) - [AT m (ONX:DNA X) + AT m (DNA Y:ONY)], where ONX:ONY is a double-stranded probe with certain interstrand zipper arrangements of disclosed monomers and 'DNA X' and 'DNA Y' are the complementary single-stranded nucleic acid targets of ONX and ONY, respectively.
  • Displace (ment): A reaction in which an atom, radical, or molecule (anionic or neutral) replaces another in a compound.
  • Double Stranded Nucleic Acid An oligonucleotide containing a region of two or more nucleotides having a double stranded motif.
  • Epitope An antigenic determinant. These are particular chemical groups or contiguous or non-contiguous peptide sequences on a molecule that are antigenic, that is, that elicit a specific immune response.
  • Fluorescence The emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength.
  • Fluorophore A functional group of a molecule which causes the molecule to be fluorescent. Typically, the functional group can absorb energy of a specific wavelength and re-emit energy at a different (but equally specific) wavelength.
  • Human epidermal growth factor receptor (Her) family A family of structurally related proteins, including at least Herl, Her2, Her3 and Her4 (aka EGFR1, EGFR2, EGFR3 and EGFR4, respectively, or ErbB-1, ErbB-2, ErbB-3 and ErbB-4, respectively).
  • Herl, Her2 and Her4 are receptor tyrosine kinases; although Her3 shares homology with Herl, Her2 and Her4, Her3 is kinase inactive.
  • Her family is p95, a truncated form of Her2 lacking portions of the Her2 extracellular domain (see, e.g., Arribas et al., Cancer Res., 71 : 1515-1519, 2011 ; Molina et al, Cancer Res., 61 :4744-4749, 2001).
  • the human epidermal growth factor family of receptors mediate cell growth and are disregulated in many types of cancer.
  • Herl and Her2 are upregulated in many human cancers, and their excessive signaling may be critical factors in the development and malignancy of these tumors. See, e.g., Herbst, Int. J. Radiat. Oncol. Biol. Phys., 59:21-6, 2004; Zhang et al., J.
  • Receptor dimerization is essential for Her pathway activation leading to receptor phosphorylation and downstream signal transduction. Unlike Herl, -3 and -4, Her2 has no known ligand and assumes an open conformation, with its dimerization domain exposed for interaction with other ligand-activated Her receptors.
  • Her2 overexpression also occurs in other cancer types, such as ovarian cancer, stomach cancer, and biologically aggressive forms of uterine cancer, such as uterine serous endometrial carcinoma. See, e.g., Santin et al., Int. J. Gynaecol. Obstet., 102 (2): 128-31, 2008.
  • Her2-containing homo- and hetero-dimers are transformation competent protein complexes. Trastuzumab, a humanized antibody that prevents Her2 homodimerization is used to treat certain
  • Her2 overexpressing cancers including breast cancer. Additionally, the level of Her2 expression in cancer tissue is predictive of patient response to Her2 therapeutic antibodies (e.g., Trastuzumab). Because of its prognostic role as well as its ability to predict response to Trastuzumab, tumors (e.g., tumors associated with breast cancer) are routinely checked for overexpression of Her2.
  • Her2 therapeutic antibodies e.g., Trastuzumab
  • Her pathway is also involved in ovarian cancer pathogenesis. Many ovarian tumor samples express all members of the Her family. Co-expression of Herl and Her2 is seen more frequently in ovarian cancer than in normal ovarian epithelium, and overexpression of both receptors correlates with poor prognosis. Preferred dimerization with Her2 (Herl/Her2, Her2/Her3) and subsequent pathway activation via receptor phosphorylation have also been shown to drive ovarian tumor cell proliferation, even in the absence of Her2 overexpression. Pertuzumab, a humanized antibody that prevents Her2 dimerization (with itself and with Her3) has been shown to provide therapeutic benefit to patients with Her2 and/orHer3 expressing ovarian cancer.
  • Herl amino acid sequence examples include NCBI/Genbank accession Nos. NP_005219.2, CAA25240.1, AAT52212.1, AAZ66620.1, BAF83041.1, BAH11869.1, ADZ75461.1, ADL28125.1, BAD92679.1, AAH94761.1.
  • Her2 amino acid sequences include NCBI/Genbank accession BAJ17684.1, P04626.1, AAI67147.1, NP_001005862.1, NP_004439.2, AAA75493.1, AAO18082.1.
  • Her3 amino acid sequences examples include NCBI/Genbank accession Nos.
  • Her4 amino acid sequences include NCBI/Genbank accession Nos., AAI43750, Q15303.1, NP_005226.1,
  • Heteroaliphatic An aliphatic group, which contains one or more atoms other than carbon and hydrogen, such as, but not limited to, oxygen, sulfur, nitrogen, phosphorus, chlorine, fluorine, bromine, iodine, and selenium.
  • homology refers to a degree of complementarity. Partial homology or complete homology can exist. Partial homology involves a nucleic acid sequence that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid.
  • Homopolymer This term refers to a polymer formed by the bonding together of multiple units of a single type of molecular species, such as a single monomer (for example, an amino acid).
  • Interstrand Zipper Nomenclature (+1/-1, etc).
  • the "interstrand zipper arrangement" nomenclature is used to describe relative arrangement between two monomers positioned on opposing strands in a duplex.
  • the number 'n' describes the distance measured in number of base pairs and has a positive value if a monomer is shifted toward the 5'-side of its own strand relative to a second reference monomer on the other strand.
  • n has a negative value if a monomer is shifted toward the 3 '-side of its own strand relative to a second reference monomer on the other strand.
  • Isolated An "isolated" microorganism (such as a virus, bacterium, fungus, or protozoan) has been substantially separated or purified away from microorganisms of different types, strains, or species. Microorganisms can be isolated by a variety of techniques, including serial dilution and culturing.
  • An "isolated” biological component such as a nucleic acid molecule, protein or organelle
  • nucleic acids and proteins that have been "isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids or proteins, or fragments thereof.
  • Leaving Group A molecular fragment that departs with a pair of electrons after heterolytic bond cleavage.
  • Leaving groups can be anions or neutral molecules.
  • Common anionic leaving groups can include halides, such as Cl ⁇ , Br ⁇ , and ⁇ , and sulfonate esters, such as para-toluenesulfonate (TsO ⁇ ), trifluoromethanesulfonate (TfO ),
  • Common neutral molecule leaving groups can include H 2 0, NH 3 , alcohols, and gases (N 2 , 0 2 , C0 2 , CO, and S0 2 ).
  • Lewis acid A chemical substance that can accept a pair of electrons from a Lewis base, B, which acts as an electron-pair donor, forming an adduct, AB as given by the following: A+:B ⁇ A— B.
  • Linker As used herein, a linker is a molecule or group of atoms positioned between two moieties.
  • Lower alkyl Any aliphatic chain that contains 1-10 carbon atoms.
  • Modified refers to an oligonucleotide that has a non-natural composition, in that it comprises one or more synthetic nucleobases which can pair with a natural base.
  • Molecule of interest or Target A molecule for which the presence, location and/or concentration is to be determined.
  • nucleobase includes naturally occuring nucleobases as well as non-natural nucleobases.
  • nucleobase encompasses purine and pyrimidine derivatives, as well as heterocyclic derivatives and tautomers thereof.
  • Nucleophile A reagent that forms a chemical bond to its reaction partner (the electrophile) by donating both bonding electrons. A molecule or ion with a free pair of electrons can act as nucleophile.
  • Nucleotide Phosphorylated nucleosides are "nucleotides,” which are the molecular building-blocks of DNA and RNA.
  • Nucleoside A glycoside of a heterocyclic base.
  • the term "nucleoside” is used broadly as to include non-naturally occurring nucleosides, naturally occurring nucleosides as well as other nucleoside analogues.
  • Illustrative examples of nucleosides are ribonucleosides comprising a ribose moiety as well as deoxyribo-nucleosides comprising a deoxyribose moiety.
  • bases of such nucleosides it should be understood that this may be any of the naturally occurring bases, e.g. adenine, guanine, cytosine, thymine, and uracil, as well as any modified variants thereof or any possible unnatural bases.
  • Oligonucleotide A plurality of joined nucleotides joined by native phosphodiester bonds.
  • An oligonucleotide is a polynucleotide of between at least 2 and about 300 nucleotides in length. Typically, an oligonucleotide is a polynucleotide of between about 5 and about 50 nucleotides.
  • An oligonucleotide analog refers to moieties that function similarly to oligonucleotides but have non- naturally occurring portions.
  • oligonucleotide analogs can contain non-naturally occurring portions, such as altered sugar moieties or inter-sugar linkages, such as a phosphorothioate oligodeoxynucleotide.
  • Functional analogs of naturally occurring polynucleotides can bind to RNA or DNA, and include locked nucleic acid (LNA) and peptide nucleic acid (PNA) molecules.
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • Pharmaceutically acceptable salts are more soluble in aqueous solutions than the corresponding free acids and bases from which the salts are produced; however, salts having lower solubility than the corresponding free acids and bases from which the salts are produced may also be formed. Pharmaceutically acceptable salts are typically
  • salts are positively charged; or the salt is counterbalanced with an inorganic acid, organic acid, or acidic amino acid if they are negatively charged.
  • Pharmaceutically acceptable salts can also be zwitterionic in form.
  • Salts can be formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and from bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine, choline, ⁇ , ⁇ '-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane, and tetramethylammonium hydroxide.
  • bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine, choline, ⁇ , ⁇ '-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane, and
  • Polypeptide A polymer in which the monomers are amino acid residues that are joined together through amide bonds. When the amino acids are oc-amino acids, either the L-optical isomer or the D-optical isomer can be used.
  • polypeptide or protein as used herein are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins.
  • polypeptide is specifically intended to cover naturally occurring proteins, as well as those which are recombinantly or synthetically produced.
  • protecting Group A moiety that can be introduced into a molecule by chemical modification of a functional group. Protecting groups often are used to protect one functional group in order to obtain chemoselectivity in a chemical reaction with a different functional group. Suitable protecting groups are well known to those of ordinary skill in the art and can include aryl groups, aliphatic groups, heteroaliphatic groups, heteroaryl groups.
  • Protein A molecule, particularly a polypeptide, comprised of amino acids.
  • purified does not require absolute purity; rather, it is intended as a relative term.
  • a purified compound is one that is isolated in whole or in part from other contaminants.
  • substantially purified peptides, proteins, conjugates, oligonucleotides, or other active compounds for use within the disclosure comprise more than 80% of all
  • the peptide, protein, conjugate or other active compound is purified to represent greater than 90%, often greater than 95% of all macromolecular species present in a purified preparation prior to admixture with other formulation ingredients.
  • the purified preparation may be essentially homogeneous, wherein other macromolecular species are not detectable by conventional techniques.
  • Quantum Yield A measure of the efficiency of the fluorescence process.
  • the "quantum yield" of a radiation-induced process indicates the number of times that a defined event occurs per photon absorbed by the system.
  • Reactive Groups Formulas throughout this application refer to "reactive groups," which can be any of a variety of groups suitable for underogoing a chemical transformation as described herein.
  • the reactive group might be an amine-reacfive group, such as an isothiocyanate, an isocyanate, an acyl azide, an NHS ester, an acid chloride, such as sulfonyl chloride, aldehydes and glyoxals, epoxides and oxiranes, carbonates, arylating agents, imidoesters, carbodiimides, anhydrides, and combinations thereof.
  • Suitable thiol-reactive functional groups include haloacetyl and alkyl halides, maleimides, aziridines, acryloyl derivatives, arylating agents, thiol-disulfide exchange reagents, such as pyridyl disulfides, TNB-thiol, and disulfide reductants, and combinations thereof.
  • Suitable carboxylate-reactive functional groups include diazoalkanes, diazoacetyl compounds, carbonyldiimidazole compounds, and carbodiimides.
  • Suitable hydroxyl-reactive functional groups include epoxides and oxiranes, carbonyldiimidazole, ⁇ '-disuccinimidyl carbonates or ⁇ -hydroxysuccinimidyl chloroformates, periodate oxidizing compounds, enzymatic oxidation, alkyl halogens, and isocyanates.
  • Aldehyde and ketone-reactive functional groups include hydrazines, Schiff bases, reductive amination products, Mannich condensation products, and combinations thereof.
  • Active hydrogen-reactive compounds include diazonium derivatives, Mannich condensation products, iodination reaction products, and combinations thereof.
  • Photoreactive chemical functional groups include aryl azides, halogenated aryl azides, benzophonones, diazo compounds, diazirine derivatives, and combinations thereof.
  • a biological specimen from a subject such as might contain genomic DNA, RNA (including mRNA), protein, or combinations thereof.
  • examples include, but are not limited to, peripheral blood, urine, saliva, tissue biopsy, surgical specimen, amniocentesis samples and autopsy material.
  • Single nucleotide polymorphism A nucleic acid sequence variation occurring when a single nucleotide in the genome (or other shared sequence) differs between members of a species or paired chromosomes in an individual. For example, two sequenced nucleic acid fragments from different individuals, AAGCCTA to AAGCTTA, contain a difference in a single nucleotide.
  • substantially complementary refers to the oligonucleotides of the disclosed methods that are at least about 50% homologous to target nucleic acid sequence they are designed to detect, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, more preferably at least about 90%, more preferably at least about 95%, most preferably at least about
  • Thermal Advantage is defined as TA (ONX:ONY) ⁇ T m (ONX:DNA X) + T m (DNA Y:ONY) - T m (ONX:ONY) - Tm (DNA X: DNA Y), where ONX:ONY is a double-stranded probe with certain interstrand zipper arrangements of disclosed monomers, ONX:DNA X and DNA Y:ONY are the duplexes between individual probe strands and nucleic acid targets, and DNA X: DNA Y is the double-stranded nuclei acid target.
  • a large, positive TA-value signifies significant potential for probe ONX:ONY to target DNA X:DNA Y.
  • Transition metal Any of the metallic elements within Groups 3 to 12 in the Periodic Table that have an incomplete inner electron shell and that serve as transitional links between the most and the least electropositive in a series of elements.
  • the probe comprises one or more pairs of monomers capable of intercalating with one or more nucleic acid targets.
  • the probe comprises one or more pairs of monomers comprising a first monomer and a second monomer arranged on opposite nucleic acid strands.
  • Certain disclosed embodiments concern a probe wherein one or more monomers are arranged in a manner that promotes thermal instability of the probe and increases the probe's ability to detect a target.
  • the disclosed probe may comprise one or more monomers capable of coupling with a nucleic acid.
  • each monomer independently may have a Formula 1 , illustrated below.
  • each Y may independently be selected from oxygen, sulfur, a triazole, oxazole, tetrazole, isoxazole, and NR b , wherein R b is selected from hydrogen, aliphatic, aryl, heteroaliphatic, and heteroaryl, and V may be selected from carbon, oxygen, sulfur, and NR b , wherein R b is as previously recited.
  • the variable "n" may range from 0 to 4; more typically, n is 1 or zero. A person of ordinary skill in the art will recognize that when n is 1 or greater, V may or may not be bound to Y, as indicated by the dashed line connecting these two variables in Formula 1.
  • R 1 may be selected from hydrogen, aliphatic, such as alkyl, more typically lower alkyl, such as methyl, ethyl, propyl, butyl, etc., alkenyl, alkynyl, aryl, aryl aliphatic, such as aryl alkyl, and a heteroatom- containing moiety.
  • the heteroatom-containing moiety may be selected from, but not limited to, ether (ROR b ), hydroxyl (R a OH), silyl ether (R a R b R c SiOR d ), phosphine (PR a R b R c ), thiol (R SH), thioether/sulfide (R a SR b ), disulfide (R a SSR b ), isothiocyanate (R NCS), isocyanate (R NCO), amine (NH 2 , NHR , NR a R b ), amide (R NR b C(0)R c ), ester (R a OC(0)R b ), halogen (I, Br, CI, F), carbonate (ROC(0)OR b ), carboxyl (R a C(0)OH), carboxylate (R a COO ), ester (R a C(0)OR b ), ketone
  • R a C(0)R b phosphate (R a OP(0)OH 2 ), phosphoryl (R a P(0)(OH) 2 ), sulfinyl (R a S(0)R b ), sulfonyl (R a S0 2 R b ), carbonothioyl (R a C(S)R b or R a C(S)H), sulfino (R a S(0)OH), sulfo (R a S0 3 H), amide (R C(0)NR b R c ), azide (N 3 ), nitrile (R CN), isonitrile (R a N + C), and nitro (R a N0 2 ).
  • R represents the remaining monomer structure, which is attached to the abovementioned functional groups at the position indicated for R 1 ; and R b , R c , and R d independently are hydrogen, aliphatic, aryl, heteroaliphatic, heteroaryl, and any combination thereof.
  • the optional linker may be selected from alkyl, amide, carbamate, carbonate, urea, and combinations thereof
  • R 2 may be selected from hydrogen, aliphatic, aryl, and any one of the heteroatom-containing moieties described herein.
  • R 2 may be selected from a protecting group known to those of ordinary skill in the art, such as, but not limited to, 4,4'-dimethoxytrityl, trityl, 9- arylthioxanthenyl, mesyl (Ms), tosyl (Ts), besoyl (Bs), trifluoromethane (CF 3 ), and trifluoromethanesulfonyl.
  • R 2 may be one or more nucleotides or monomers.
  • R 3 typically may be a heteroatom-containing functional group.
  • the heteroatom may be selected from phosphorous, sulfur, nitrogen, oxygen, selenium, and/or a metal.
  • Certain disclosed embodiments utilize R 3 substituents having a formula
  • R 3 is selected from oxygen, sulfur, NR b where R b is selected from hydrogen, aliphatic, aryl, heteroaliphatic, heteroaryl, and W is selected from phosphorus, SH, or SeH.
  • R 3 substituents include, without limitation, the following:
  • R 3 has a formula
  • W is phosphorus
  • each Z independently is selected from ether, thioether, hydroxyl, and NR b 2 .
  • R 3 may be
  • R 4 may be selected from any natural or non-natural nucleobase.
  • R 4 is a natural nucleobase selected from uracil, adenine, thymine, cytosine, guanine.
  • a person of ordinary skill in the art will recognize that R 4 may also be any non-natural, or synthetically developed nucleobase including those presently known or developed in the future.
  • R 4 may be selected from C-5 functionalized pyrimidines, C6-functionalized pyrimidines, C7-functionalized 7-deazapurines, C8 -functionalized purines, 2,6-diaminopurine, 2- thiouracil, 4-thiouracil, deoxyinosine and 3-(2'-deoxy- -D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimdine- 2-(3H)-one.
  • R 5 may be an intercalator capable of intercalating within a nucleic acid.
  • R 5 may be any moiety capable of intercalating with single stranded nucleic acids, double stranded nucleic acids, and/or triple stranded nucleic acids.
  • R 5 may be a planar moiety capable of maintaining a flat orientation when inserted into a nucleic acid.
  • R 5 may be a hydrocarbon selected from pyrene, coronene, perylene, anthracene, naphthalene, and functionalized derivatives thereof; or an aromatic heterocycle, such as a porphyrin, a nucleobase (such as pyrimidines, purines, size-expanded nucleobases), a metal chelator (such as phenanthroline, DPPZ), an azapyrene, thiazole orange, ethidium, a diazobenzene, an indole, a pyrrole, benzimidizoles, and modified analogs thereof.
  • R 5 may be modified to include various aliphatic, aryl, or heteroatom-containing functional groups.
  • Particular disclosed embodiments concern a probe comprising one or more monomers selected from any one of Formulas 3-5.
  • the probe may comprise one or more monomers having any one of Formulas 6 and 7.
  • B may be selected from uracil, adenine, thymine, guanine, cytosine and 2-thiouracil with or without common protecting groups
  • R 5 may be selected from napth-2-yl, pyren-l-yl, coronen-l-yl, CH 2 -pyren-l-yl, CH 2 -coronen-l -yl, CO-pyren-l-yl, COCH 2 -pyren- 1 -yl, CH 2 -perylen-3-yl; CH 2 -l-(7-neopentylpyrenyl), CH 2 -l-(6- bromopyrenyl), CH 2 -l -(8-bromopyrenyl), CH 2 -l-(8-methylpyrenyl), CH 2 -l-(7-tert-butyl-3-
  • Particular disclosed embodiments concern using one or more monomers that do not participate in base pairing when either forming the probe or when the probe is reacted with a target.
  • the monomers that do not participate in base pairing are herein referred to as "bulge monomers.”
  • the bulge monomers may comprise one or more aliphatic, abasic sites, heteroalkyl groups, natural/modified nucleotides, and combinations thereof.
  • One or more of the bulge monomers may be included in the probe.
  • the single-stranded probe precursor may comprise one or more monomers arranged sequentially or in a manner wherein one or more natural or non-natural nucleotides are located between two or more monomers.
  • a single-stranded probe may serve as a precursor to a duplex comprising one or more of the disclosed monomers, or it may be used in the disclosed method discussed herein.
  • Certain disclosed embodiments concern a single-stranded probe having a general Formula 9, illustrated below.
  • the disclosed single-stranded probe precursor may comprise locked monomers, unlocked monomers, or combinations thereof.
  • B ⁇ , B m . s may be any natural or non-natural nucleotide presently known or discovered in the future, wherein m-s may range from zero to about 28.
  • g, h, i and j may range from 0 to 10, more typically 0 to 5, and even more typically 0 to 1.
  • Each X independently may be selected from any of the disclosed monomers and A may be a Watson-Crick base pairing nucleotide, or derivative thereof, which is capable of coupling with a complementary Watson-Crick base pairing nucleotide, or derivative thereof, in a target.
  • each X independently may be a monomer having any one of formulas 1 -7 and A may be selected from uracil, adenine, guanine, thymine, cytosine, and derivatives thereof.
  • the single-stranded probe precursor may have a
  • One or more bulge monomers also may be inserted at any position within a single stranded or double stranded probe.
  • Probes may be additionally modified with one or more non-pairing modifications (a non-pairing modification is anything that can be incorporated internally within an oligonucleotide), which serves to decrease the thermostability of the probe, which facilitates the dsDNA-recognition reaction.
  • non-pairing modification is anything that can be incorporated internally within an oligonucleotide
  • the probe may be a duplex comprising two strands of oligonucleotides comprising one or more pairs of the disclosed monomers.
  • the one or more pairs of the disclosed monomers are arranged in a manner that substantially decrease the duplex's thermal stability, thereby providing the probe with the ability to bind to the target.
  • arranging the monomers in the particular manner disclosed herein provides the probe with sufficient energy to dissociate (or denature) into two strands that then couple with the target (FIG. 1).
  • the probe may comprise more than one pairs of monomers; for example, the probe may comprise anywhere from 1 pair of monomers to about 5 pairs of monomers.
  • a pair of monomers can comprise two monomers, having any of the formulas disclosed herein, that are located on opposite strands of the probe (e.g. opposite strands of the duplex).
  • a first monomer may be positioned at any location on one of the probe strands, with the second monomer of the pair being positioned at a particular location relative to the first monomer on the other probe strand.
  • Certain disclosed embodiments concern a probe having one or more pairs of monomers arranged in a (+/-)n zipper arrangement, wherein n can range from 0 to about 10; more typically from 0 to about 3; even more typically from at least 1 to about 2.
  • Particular disclosed embodiments concern a probe having at least one pair of monomers arranged in a +n zipper arrangement. Without being limited to a particular theory of operation, it currently is believed that certain arrangements of the interstrand monomers result in destabilization of the probe, such as illustrated in FIGS. 1-2.
  • the pair of monomers may be arranged in a -n zipper arrangement
  • Exemplary embodiments of the disclosed probe typically comprise a pair of monomers comprising a first monomer and a second monomer arranged in a (+1) interstrand zipper arrangement.
  • Other exemplary embodiments concern a probe comprising two pairs of monomers, with the first monomer and the second monomer of each pair being arranged in a (+1) interstrand zipper arrangement and each pair of monomers being separated by at least 0 to about 10 natural or non-natural nucleotides or bulge monomers.
  • the disclosed probe may have a Formula 10, illustrated below.
  • the disclosed probe may comprise locked monomers, unlocked monomers, and combinations thereof.
  • Bi, B 2 ...B m-i may be any natural or non-natural nucleotide, presently known or discovered in the future, wherein m-s may range from zero to about 28.
  • g, h, i and j may range from 0 to 1,000, such as 0 to 900, such as 0 to 800, such as 0 to 700, such as 0 to 600, such as 0 to 500, such as 0 to 400, such as 0 to 300, such as 0 to 200, such as 0 to 100, such as 0 to 50, typically 0 to 10, and even more typically 0-5 X may be selected from any of the disclosed monomers and A may be a Watson-Crick base pairing nucleotide, or derivative thereof, or another disclosed monomer, which is capable of coupling with a complementary Watson-Crick base pairing nucleotide, or derivative thereof, in a target.
  • X may be a monomer having any one of Formulas 1-7, and A may be selected from nucleotides with uracil, adenine, guanine, thymine or cytosine nucleobases, or nucleotides with pseudocomplementary nucleobases (e.g., 2-thiouracil, 2,6-diaminopurine, inosine, 3- pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one).
  • C may be any natural or non-natural nucleotide, presently known or discovered in the future, that is capable of Watson-Crick base pairing with any one of B B 2 ... m . s .
  • B and “C” also can be pseudocomplementary base pairs that do not form strong (or any base pairs);
  • P may be selected from any of the disclosed monomers, and D may be a Watson-Crick base pairing nucleotide, or derivative thereof, or another disclosed monomer, which is capable of coupling with a complementary Watson-Crick base pairing nucleotide, or derivative thereof, in a target.
  • P may be a monomer having any one of Formulas 1-7, and D may be selected from uracil, adenine, guanine, thymine, and cytosine.
  • FIGS. 3A and B illustrates the concept of using bulge monomers.
  • green denotes disclosed the intercalator-functionalized monomers; red represents a non- nucleosidic linker.
  • One or more bulge monomers can be added to any of the embodiments defined by the above formula.
  • Exemplary embodiments of the disclosed probe are provided in Table 1.
  • a person of ordinary skill in the art will recognize that any of the disclosed embodiments of the probe can be made from two single-stranded precursors that are combined (hybridized) to form the probe.
  • the embodiments disclosed in Table 1 concern a probe wherein X is a monomer disclosed herein and R 4 is defined as a uracil or thymine nucleobase; however a person of ordinary skill in the art would recognize that any of the disclosed monomers may be used.
  • Table 2 Additional exemplary embodiments are disclosed in Table 2.
  • the embodiments disclosed in Table 2 concern a probe wherein the monomer in each strand (e.g. the strands designated as 5' and 3') may have any one of formulas 1-7, wherein the R 4 moiety is selected from adenine (A), cytosine (C), guanine (G), uracil (U), or thymine (T).
  • A adenine
  • C cytosine
  • G guanine
  • U uracil
  • T thymine
  • Tables 4-6 outline additional working embodiments of certain exemplary probes targeting different DNA regions, i.e., second insulin [INSB], PPAR gamma and CEBP promotors.
  • INLB second insulin
  • PPAR gamma PPAR gamma
  • CEBP promotors PPAR gamma
  • the target may be a nucleic acid, such as, but not limited to, single-stranded DNA (and derivatives thereof), double-stranded DNA (and derivatives thereof), and any combinations thereof.
  • Particular disclosed embodiments concern targeting isosequential (relative to the probe) double stranded DNA target regions, including: stems of molecular beacons, target regions embedded within PCR amplicons, target regions embedded within circular or linearized plasmids, target regions embedded within genomic DNA (crude, purified, cell culture, in vivo, embryos, etc.), target regions embedded within microorganisms, and the like.
  • the target may be selected by identifying an RNA target, such as those pursued in antisense/siRNA/anti-miRNA clinical trials and pre-clinical trials (e.g. those used in modulation of gene expression and/or identification of biomarkers) and design a target comprising the corresponding DNA to this particular RNA target.
  • specific targets include linearized plasmids (e.g. against T7 promotor, as illustrated by FIGS. 18-20); circular plasmids (e.g. against insulin B promotor in circular plasmids, as illustrated by FIGS. 21-23);
  • genomic DNA genomic DNA
  • structured dsDNA targets FIGGS 24-26 and FIGS 30-42.
  • a target nucleic acid sequence can vary substantially in size. Without limitation, the nucleic acid sequence can have a variable number of nucleic acid residues. For example a target nucleic acid sequence can have at least about 2 nucleic acid residues, typically at least about 10 nucleic acid residues, or at least about 20, 30, 50, 100, 150, 500, 1000 residues.
  • a protein is produced by a target nucleic acid sequence (e.g., genomic target nucleic acid sequence) associated with a neoplasm (for example, a cancer).
  • a target nucleic acid sequence e.g., genomic target nucleic acid sequence
  • Numerous chromosome abnormalities have been identified in neoplastic cells, especially in cancer cells, such as B cell and T cell leukemias, lymphomas, breast cancer, colon cancer, neurological cancers and the like. Therefore, in some examples, at least a portion of a protein is produced by a nucleic acid sequence (e.g., genomic target nucleic acid sequence) that is amplified or deleted in at least a subset of cells in a sample.
  • Oncogenes are known to be responsible for several human malignancies. For example, chromosomal rearrangements involving the SYT gene located in the breakpoint region of chromosome 18ql 1.2 are common among synovial sarcoma soft tissue tumors. The t(18ql 1.2) translocation can be identified, for example, using the disclosed probe.
  • a protein produced from a nucleic acid sequence e.g., genomic target nucleic acid sequence
  • this type of target may also be detected, identified, the expression of the gene reduced and/or the gene modified using disclosed probe embodiments.
  • the pl6 region (including D9S 1749, D9S 1747, pl6(INK4A), pl4(ARF), D9S 1748, pl5(INK4B), and D9S 1752) located on chromosome 9p21 is deleted in certain bladder cancers.
  • Chromosomal deletions involving the distal region of the short arm of chromosome 1 that encompasses, for example, SHGC57243, TP73, EGFL3, ABL2, ANGPTL1, and SHGC-1322
  • the pericentromeric region e.g., 19pl3-19ql3 of chromosome 19
  • MAN2B 1, ZNF443, ZNF44, CRX, GLTSCR2, and GLTSCR1 are characteristic molecular features of certain types of solid tumors of the central nervous system.
  • Target proteins that are produced by nucleic acid sequences (e.g., genomic target nucleic acid sequences), which have been correlated with neoplastic transformation and which are useful in the disclosed methods, also include the EGFR gene (7pl2; e.g., GENBANKTM Accession No. NC_000007, nucleotides
  • the C-MYC gene (8q24.21 ; e.g., GENBANKTM Accession No. NC_000008, nucleotides 128817498-128822856), D5S271 (5pl5.2), lipoprotein lipase (LPL) gene (8p22; e.g., GENBANKTM Accession No. NC_000008, nucleotides 19841058-19869049), RB I (13ql4; e.g., GENBANKTM Accession No. NC_000013, nucleotides 47775912-47954023), p53 (17pl3.1 ; e.g., GENBANKTM Accession No. NC_000017, complement, nucleotides 7512464-7531642)), N-MYC (2p24; e.g., GENBANKTM Accession No. NC_000002, complement, nucleotides
  • nucleotides 29269144-29997936 Ig heavy chain, CCND1 (1 lql3; e.g., GENBANKTM Accession No. NC_000011, nucleotides 69165054..69178423), BCL2 (18q21.3; e.g., GENBANKTM Accession No. NC_000018, complement, nucleotides 58941559-59137593), BCL6 (3q27; e.g., GENBANKTM Accession No. NC_000003, complement, nucleotides 188921859-188946169), MALF1, API (lp32- p31 ; e.g., GENBANKTM Accession No. NC_000001, complement, nucleotides 59019051-59022373), TOP2A (17q21-q22; e.g., GENBANKTM Accession No. NC_000017, complement,
  • TMPRSS 21q22.3; e.g., GENBANKTM Accession No.
  • NC_000021, complement, nucleotides 41758351-41801948 ERG (21q22.3; e.g., GENBANKTM Accession No. NC_000021, complement, nucleotides 38675671-38955488); ETV1 (7p21.3; e.g., GENBANKTM Accession No. NC_000007, complement, nucleotides 13897379-13995289), EWS (22ql2.2; e.g., GENBANKTM Accession No. NC_000022, nucleotides 27994271-28026505); FLU (I lq24.1-q24.3; e.g., GENBANKTM Accession No. NC_000011, nucleotides
  • PAX3 (2q35-q37; e.g., GENBANKTM Accession No. NC_000002, complement, nucleotides 222772851-222871944
  • PAX7 Ip36.2-p36.12; e.g., GENBANKTM Accession No. NC_000001, nucleotides 18830087-18935219
  • PTEN 10q23.3; e.g., GENBANKTM Accession No. NC_000010, nucleotides 89613175-89716382
  • AKT2 (19ql3.1-ql3.2; e.g.,
  • REL 2pl3-pl2; e.g., GENBANKTM Accession No. NC_000002, nucleotides 60962256-61003682
  • CSF1R 5q33-q35; e.g., GENBANKTM Accession No. NC_000005, complement, nucleotides 149413051-149473128).
  • a target protein is selected from a virus or other microorganism associated with a disease or condition. Detection of the virus- or microorganism-derived target nucleic acid sequence (e.g., genomic target nucleic acid sequence) in a cell or tissue sample is indicative of the presence of the organism.
  • the disclosed probe may be used to detect and/or identify these types of targets.
  • a gene encoding a critical enzyme for the survival of a microorganism can be targeted by disclosed probe embodiments, which can cause the death of the microorganism.
  • the target protein can be selected from the genome of an oncogenic or pathogenic virus, a bacterium or an intracellular parasite (such as Plasmodium falciparum and other Plasmodium species, Leishmania (sp.), Cryptosporidium parvum, Entamoeba histolytica, and Giardia lamblia, as well as Toxoplasma, Eimeria, Theileria, and Babesia species).
  • an oncogenic or pathogenic virus such as Plasmodium falciparum and other Plasmodium species, Leishmania (sp.), Cryptosporidium parvum, Entamoeba histolytica, and Giardia lamblia, as well as Toxoplasma, Eimeria, Theileria, and Babesia species).
  • the target protein is produced from a nucleic acid sequence (e.g., genomic target nucleic acid sequence) from a viral genome.
  • a nucleic acid sequence e.g., genomic target nucleic acid sequence
  • Exemplary viruses and corresponding genomic sequences may selected from the following (GENBANKTM RefSeq Accession No.
  • human adenovirus A (NC_001460), human adenovirus B (NC_004001), human adenovirus C (NC_001405), human adenovirus D (NC_002067), human adenovirus E (NC_003266), human adenovirus F (NC_001454), human astrovirus (NC_001943), human BK polyomavirus (VOl 109; GL60851) human bocavirus (NC_007455), human coronavirus 229E (NC_002645), human coronavirus HKU1 (NC_006577), human coronavirus NL63 (NC_005831), human coronavirus OC43 ( NC_005147), human enterovirus A (NC_001612), human enterovirus B (NC_001472), human enterovirus C (NC_001428), human enterovirus D (NC_001430), human erythrovirus V9 (NC_004295), human foam
  • NC_009333 human immunodeficiency virus 1 (NC_001802), human immunodeficiency virus 2 (NC_001722), human metapneumovirus (NC_004148), human papillomavirus- 1 (NC_001356), human papillomavirus- 18 (NC_001357), human papillomavirus -2 (NC_001352), human papillomavirus -54 (NC_001676), human papillomavirus -61 (NC_001694), human
  • NC_004104 human papillomavirus RTRX7 (NC_004761), human papillomavirus type 10 (NC_001576), human papillomavirus type 101 (NC_008189), human papillomavirus type 103 (NC_008188), human papillomavirus type 107 (NC_009239), human papillomavirus type 16 (NC_001526), human papillomavirus type 24 (NC_001683), human papillomavirus type 26 (NC_001583), human papillomavirus type 32 (NC_001586), human papillomavirus type 34 (NC_001587), human papillomavirus type 4 (NC_001457), human papillomavirus type 41 (NC_001354), human papillomavirus type 48 (NC_001690), human papillomavirus type 49 (NC_001354), human papilloma
  • the target protein is produced from a nucleic acid sequence (e.g., genomic target nucleic acid sequence) from an oncogenic virus, such as Epstein-Barr Virus (EBV) or a Human Papilloma Virus (HPV, e.g., HPV16, HPV18).
  • a nucleic acid sequence e.g., genomic target nucleic acid sequence
  • EBV Epstein-Barr Virus
  • HPV Human Papilloma Virus
  • the target protein produced from a nucleic acid sequence is from a pathogenic virus, such as a Respiratory Syncytial Virus, a Hepatitis Virus (e.g., Hepatitis C Virus), a Coronavirus (e.g., SARS virus), an Adenovirus, a Polyomavirus, a Cytomegalovirus (CMV), or a Herpes Simplex Virus (HSV).
  • a pathogenic virus such as a Respiratory Syncytial Virus, a Hepatitis Virus (e.g., Hepatitis C Virus), a Coronavirus (e.g., SARS virus), an Adenovirus, a Polyomavirus, a Cytomegalovirus (CMV), or a Herpes Simplex Virus (HSV).
  • Other targets contemplated by the present disclosure include Herl, Her2, Her3 and Her4 (e.g. EGFR1, EGFR2, EGFR3 and EGFR4, respectively, or
  • Particular disclosed embodiments concern a method of making the disclosed monomers.
  • the method may concern synthesizing locked monomers (e.g.
  • the method may concern synthesizing unlocked monomers (e.g. monomers having Formulas 1, 2, or 4).
  • the monomer may be a locked monomer, which may be synthesized from a precursor 2 using the synthetic sequence illustrated in Scheme 1.
  • precursor nucleoside 2 may be converted to an N2'-functionalized locked nucleic acid using a number of chemical transformations.
  • Treating precursor nucleoside 2 may be converted to azide 4 using methods known to those of ordinary skill in the art, such as by converting the YH group of precursor nucleoside 2 to a leaving group, such as a mesylate, triflate, and/or tosylate, and displacing the leaving group using a nucleophilic azide compound, such as sodium azide.
  • Azide 4 can then be converted to locked nucleoside 6 using a tandem Staudinger reaction (iminophosphorane formation)/intramolecular nucleophilic substitution sequence. Protection and protecting group manipulation of N2' nucleoside 6 followed by deprotection ultimately provides locked nucleoside 10 in a number of steps.
  • the locked nucleoside 10 may be converted to a monomer suitable for implementation into the disclosed probe.
  • Scheme 2 illustrates the conversion of locked nucleoside 10 to such a monomer.
  • the locked nucleoside 10 can be converted to protected nucleoside 20.
  • N2' functionalization of protected nucleoside 20 using a variety of substituents can carried out using conditions known to a person of ordinary skill in the art as being suitable for coupling an amine with various functional groups.
  • N2' functionalized nucleoside 22 can comprise an optional linker and an R 5 moiety selected from any of the R 5 moieties disclosed herein.
  • a monomer 24 can be made.
  • Monomer 24 is suited for further incorporation into a nucleic acid sequence.
  • Nucleoside 34 was reacted with trifluoromethanesulfonic anhydride to facilitate formation of the 02'-triflate 36 which was subsequently without intermediate purification treated with sodium azide and 15-crown-5 in anhydrous DMF to afford azide 38.
  • IR spectroscopy verified the presence of the azide functionality (sharp band at 2115 cm "1 ) and provided along with NMR and HRMS-MALDI, evidence for the proposed structure of azide 38.
  • Azide 38 was converted into the desired bicyclic nucleoside 40 in 80% yield using a one -pot tandem Staudinger/intramolecular nucleophilic substitution reaction.
  • Protecting nucleoside 40 with a trifluoroacetyl group using trifluoroacetic anhydride in anhydrous dichloromethane and anhydrous pyridine facilitated formation of nucleoside 42 in 70% yield.
  • Nucleoside 42 was subsequently subjected to benzylic ether cleavage conditions using BC1 3 in anhydrous dichloromethane affording debenzylated nucleoside 44 in yields of 65-87%.
  • Debenzylation was followed by exchanging the methanesulfonyl protecting group at C-5' with a benzoyl protecting group.
  • the reaction was carried out under anhydrous conditions using sodium benzoate and 15-crown-5 in DMF affording nucleoside 46 in isolated yields of 70 - 83%.
  • Subjecting nucleoside 46 to sodium hydroxide in water and 1,4-dioxane cleaved both the 5 '-benzoyl and trifluroacetic acid protecting groups. Purification of the amino diol afforded target nucleoside 48 in 60-80% isolated yield.
  • the hydroxyl group of nucleoside 48 was subsequently protected at the 5'- position by 4,4'-dimethoxytrityl (DMTr) to afford the DMTr-protected nucleoside 50.
  • DMTr 4,4'-dimethoxytrityl
  • Scheme 4 illustrates an exemplary method of converting a DMTr-protected nucleoside 50 to different exemplary embodiments of the disclosed monomers.
  • monomer 51W was synthesized from nucleoside 50 using 9- fluorenylmethoxycarbonyl chloride (Fmoc-Cl) in anhydrous pyridine at 0°C for 6h, and isolated in 51 % yield.
  • Nucleoside 51 W was converted into the corresponding amidite 52W for use during oligonucleotide synthesis using 2-cyanoethyl-A ⁇ A ⁇ -(diisopropyl)-phosphoramidochloridite and diisopropylethylamine in anhydrous dichloromethane.
  • Pyrenylmethyl derivative 51X was synthesized from nucleoside 50 via reductive amination, using pyrene carboxaldehyde and sodium triacetoxy borohydride in 1,2-dichloroethane at room temperature for 24h, and was isolated in 60% yield.
  • the funcfionalized nucleoside 51X was converted into the corresponding amidite 52X for use during oligonucleotide synthesis, using 2- cyanoethyl-A ⁇ A ⁇ -(diisopropyl)-phosphoramidochloridite and 20% diisopropylethylamine in anhydrous dichloromethane.
  • the amide bond of 51Y was formed via a l-ethyl-3-(3-dimethyl-amino-propyl)- carbodiimide hydrochloride (EDC HCl) mediated coupling.
  • EDC HCl carbodiimide hydrochloride
  • the reaction was performed by dissolving bicyclic nucleoside 50 in anhydrous dichloromethane and adding pyrenecarboxylic acid and EDC HCl. The reaction mixture was stirred at room temperature for 45h. The reaction mixture was subsequently subjected to standard aqueous workup and purification, isolating nucleoside 51Y in 64% yield.
  • Nucleoside 51Y was converted into the corresponding phosphoramidite 52Y using 2- cyanoethyl-A'.A ⁇ '-idiisopropy ⁇ -phosphoramidochloridite and 20% diisopropylethylamine in anhydrous dichloromethane. Similar
  • bicyclic nucleoside 50 was converted to monomer 51Z by adding
  • nucleoside 51Z was converted into the corresponding phosphoramidite 52Z using 2- cyanoethyl-A'.A ⁇ '-idiisopropy ⁇ -phosphoramidochloridite and diisopropylethylamine in anhydrous dichloromethane.
  • R 4 moiety is selected to be thymine.
  • 05'-tritylated bicyclic nucleoside 54 which may be obtained from commercially available diacetone-oc-D-glucose in 5% overall yield over seventeen steps involving eight chromatographic purification steps, was used as a suitable starting material for the synthesis of N2'-functionalized 2'-amino-a-L-LNA phosphoramidites 58Q-58Z.
  • the disclosed monomers are selected to probe the available structural space in nucleic acids and fall into two groups based on the nature of the N2' -moiety (e.g. monomers with small non-aromatic units
  • nucleoside 56V Treatment of amino alcohol 54 with slight excess of acetic anhydride followed by selective 03'- deacylation using dilute methanolic ammonia provided nucleoside 56V in excellent 88% yield over two steps.
  • a HATU-mediated coupling procedure were used in order to improve the yield of 56X to 90%.
  • NMR signals of the exchangeable 3'-OH protons upon D 2 0 addition confirmed the N2'-functionalized constitution of nucleosides 56S-56Z, which subsequently were converted to the corresponding phosphoramidites 58S-58Z using 2-cyanoethyl A ⁇ ,A ⁇ '-(diisopropyl)-phosphoramidochloridite and diisopropylethyl amine (Hunig's base). While amidites 58S-58Y were obtained in good to excellent yields (60-90 %), 58Z is obtained in 36% yield.
  • the yield of 58X was improved using bis-(A ⁇ ,A ⁇ -diisopropylamino)-2-cyanoethoxyphosphine in dichloromethane with diisopropylammonium tetrazolide as an activator.
  • unlocked monomers and a method of making these monomers.
  • the unlocked monomers may be made in approximately three steps from a particular compound.
  • a particular disclosed embodiment of a method of making the unlocked monomers is illustrated in Scheme 6.
  • bicyclic nucleoside 60 may be converted to nucleoside 62 via methods known to those of ordinary skill in the art, such as Lewis acid-mediated ring-opening and/or heat-catalyzed nucleophilic addition.
  • an alcohol, thiol, or amine may be used to open the ring illustrated in Scheme 6.
  • These reagents may be combined with a Lewis acid, such as a borane, and heat to facilitate functionalization and conversion of the bicyclic nucleoside 60 into nucleoside 62.
  • nucleoside 62 can be converted to protected nucleoside 64, which may then be further protected to produce monomer 66.
  • nucleoside 62 may be converted to a compound having a di-functionalized Y2' moiety at the C2' position, such as compound 68, which can be made by methods known in the art, such as via ring-opening and functional group manipulation.
  • compound 68 can be exposed to coupling conditions known to those of ordinary skill in the art, such as, but not limited to activated couplings and base mediated couplings.
  • activating agents such as A ⁇ -ethyl-A ⁇ '-(3-dimethylaminopropyl)carbodiimide (EDC), l-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDCI), dicyclohexylcarbodiimide (DCC), carbonyl diimidazole (CDI), 1 -hydroxybenzotriazole (HOBt), l-hydroxy-7-aza-benzotriazole (HO At), and o-(7-azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) may be used in the activated coupling.
  • coupled nucleosides may also be obtained by treating nucleoside 68 with a base, such as an aliphatic amine base (e.g.
  • Monomer 70 is made via base-mediated functionalization.
  • an optional linker is inserted between Y and R 5 and/or R 6 .
  • Sulfur analogs can be used in the general approach illustrated in Scheme 6.
  • An example of this approach is illustrated below in Scheme 6A where a sulfur nucleophile is introduced using a nucleophile R 5 YH and a base (e.g., NaH).
  • the monomer may be synthesized using the methods illustrated in Scheme 7, below.
  • Scheme 7 A person of ordinary skill in the art will recognize that the methods of Scheme 7 are exemplary only and are not intended to be limiting.
  • Scheme 7A According to Scheme 7 A, 2,2'-anhydrouridine 70 is treated with neat phenols, such as 2- naphtol and 1-pyrenol, to afford 02'-arylated uredines 72W and 72X in 25% and 44% yield, respectively.
  • This method was adapted in order to obtain nucleosides 72Y and 72Z by treating bicyclic nucleoside 70 with tris(pyrene-l-ylmethyl) or tris(coronene-l-ylmethyl) borate - generated in situ upon addition of pyren-1 -ylmethanol or coronen-1 -ylmethanol to borane. This modification afforded nucleosides 72 Y and 72Z in reproducible yields.
  • nucleosides 74W-74Z in 47-78% yield, which upon treatment with 2-cyanoethyl-A ⁇ ,A ⁇ - diisopropylchlorophosporamidite (PCl-reagent) and diisopropylethylamine (Hunig's base) provided target phosphoramidites 76W-76Z in 74-78% yield (Scheme 7).
  • Scheme 7B provides additional examples of monomers obtained via this method.
  • Scheme 7B Other particular disclosed embodiments of making the disclosed monomers are illustrated in Schemes 8 and 9. With reference to Scheme 8, and concerning the exemplary conversion of 128 to
  • certain embodiments used 02' alkylation, typically using a haloalkane and base, such as arylmethylhalide and sodium hydride.
  • methylamine nucleoside 80 (made from 5'-0- dimethoxytrityl-2,2'-anhydrouridine in -73% yield over three steps) may undergo direct N2'- alkylation using pyren- 1 -ylmethyl chloride afforded the desired product 82Q in 46% yield.
  • triacetoxyborohydride or sodium cyanoborohydride failed to afford 82Q in acceptable yields due to prominent formation of the corresponding cyclic N2',03' -hemiaminal ether. While formation of this byproduct was avoided by prior protection of the 03'-position of 80 as a TBDMS-ether, the increased steric bulk resulted in low yields during the subsequent reductive amination.
  • HC ⁇ CCH 2 ZnBr (generated in situ from propargyl bromide and activated zinc in THF) to pyrene- 1 - carboxaldehyde, followed by deoxygenation of the resultant homopropargyl alcohol using trifluoroboron etherate and triethylsilane, afforded 94 in 31 % yield.
  • 05' -protection hereof provided 264, which upon treatment with a sulphur source produced 2-thiouracil derivative 266.
  • Disclosed embodiments concern a method of making a probe capable of recognizing a target, particularly a double stranded DNA target.
  • the probe is a nucleic acid duplex that comprises at least one pair of monomers that are capable of reducing the thermal stability of the duplex and thus promote dissociation of two strands of the duplex.
  • the probe may be able to identify an isosequential nucleic acid sequence.
  • the probe may be made by first identifying a desired target, such as a particular nucleic acid sequence, and then constructing the probe to be a complement to such sequence by developing a probe having nucleotides capable of Watson-Crick base pairing with the target.
  • the probe may be modified with at least one pair of monomers, whereas the isosequential target lacks such a modification.
  • the probe is constructed by converting one or more of the monomers disclosed herein to an oligonucleotide, wherein the monomer is coupled with one or more natural or non-natural nucleobases to form a modified oligonucleotide.
  • the probe may be designed as a double stranded DNA probe (e.g. monomers and natural and/or non-natural nucleobases bound together via phosphate moieties), a double stranded phosphorothioate-DNA probe (e.g.
  • RNA probe monomers and natural and/or non-natural nucleobases bound together via one or more phosphorothioate moieties
  • a triazole-linked DNA or RNA probe an unmodified RNA probe, modified RNA probe and/or other non-natural DNA/RNA strands now known or hereafter discovered or made.
  • Scheme 14 illustrates a particular disclosed embodiment of a method for making the probe.
  • a monomer may be incorporated into an oligonucleotide using methods known to a person of ordinary skill in the art, such as by using a nucleic acid synthesizer.
  • monomer 110 may be converted into oligonucleotide 112, wherein the wavy lines indicate the position at which one or more natural or non-natural nucleotides and/or additional identical or different monomers may be coupled.
  • the transformation illustrated in Scheme 14 can be obtained by any methods known to those of ordinary skill in the art, such as by using an activator, such as an imidazole, triazole, or tetrazole compound, and an oxidant, such as iodine or a peroxide compound.
  • an activator such as an imidazole, triazole, or tetrazole compound
  • an oxidant such as iodine or a peroxide compound.
  • Particular embodiments utilize dicyanoimidazole as the activator.
  • peroxide compounds include, but are not limited to, hydrogen peroxide or tert -butyl hydrogen peroxide.
  • Particular disclosed embodiments may concern making the disclosed probe according to the method illustrated in Scheme 16.
  • intercalator-functionalized phosphoramidites 120W-120Z, 120Q- 120V, 122W-122Z, and 124W-124Y were incorporated into oligonucleotides via machine - assisted solid-phase DNA synthesis using an activator, such as an activator selected from 4,5- dicyanoimidazole and 5-(bis-3,5-trifluoromethylphenyl)-li/-tetrazole, for time periods ranging from about 1 minute to about 40 minutes; more typically from about 10 minutes to about 35 minutes.
  • Particular embodiments concern probes simultaneously comprising one or more of the disclosed monomers and one or more non-pairing or bulge monomers.
  • non-pairing or bulge monomers 402-4, 402-9 and 402-N were incorporated into
  • oligonucleotides via machine-assisted solid-phase DNA synthesis as recommended by commercial vendors and/or in an equivalent manner as described for the disclosed intercalator-functionalized monomers.
  • denaturation curves may display sigmoidal monophasic transitions, such as those exemplary embodiments illustrated in FIGS. 4-5. Changes in thermal denaturation temperatures (r m -values) of modified duplexes are discussed relative to T m - values of unmodified reference duplexes, unless otherwise mentioned.
  • Certain embodiments entail single-stranded probes comprising the disclosed monomers. It currently is believed that certain disclosed monomers result in significantly increased thermal affinity toward single-stranded nucleic acid targets, more commoncly single-stranded DNA. For example, 9- mer oligonucleotides (ONs), which are modified with locked monomers 126W-Z, display extraordinary thermal affinity toward complementary DNA relative to unmodified ONs (AT m up to +19.5 °C, Table 8).
  • ONs 9- mer oligonucleotides
  • the rigid 2'-N- alkanoyl linker positions the intercalator in an unsuitable position for affinity-enhancing intercalation and/or that increased solvation of the linker stabilizes the single-stranded state rendering hybridization less energetically favorable.
  • aAT m change in T m values relative to unmodified reference duplex D1:D2 (T m - 29.5 °C); see Table 8 for experimental conditions.
  • ONX:ONY is a duplex with an interstrand monomer arrangement
  • 'DNA X' and 'DNA Y' are the complementary DNA of ONX and ONY, respectively.
  • DA ⁇ 0 °C for additive impacts, DA » 0 °C for more-than-additive impacts, and DA « 0 °C for less-than-additive impacts see also definitions.
  • Double-stranded probes may display a largely positive TA (or largely negative DA). This energy difference between probe-target duplexes on one side and double-stranded nucleic acid targets (more commonly dsDNA) and probes on the other side, may provide the driving force for recognition of double-stranded target regions (more commonly dsDNA target regions), via the method shown in FIG. 1.
  • Particular embodiments entail double-stranded probes with +1 interstrand zipper arrangements (see also definitions) of disclosed monomers as shown in Tables 1 1 and 12, which display significant dsDNA-targeting potential as evidenced by the highly negative DA-values (DA between -40 °C and -12 °C).
  • probes with +2 zipper arrangements of intercalator-modified monomers which also display negative DA-values, although the values are generally less prominent than probes with +1 zipper monomer arrangements.
  • Probes with other interstrand zipper arrangements e.g., +4, -1 and -3) display less regular and/or prominent dsDNA-targeting potential as indicated by DA-values ranging from moderately negative to moderately positive (DA between -8.5 °C and +4 °C).
  • probes with +1 interstrand arrangements of disclosed monomers display significant potential for targeting of isosequential dsDNA regions, and enable targeting of isosequential double- stranded nucleic acid regions, more commonly double-stranded DNA, via the method outlined in FIG. 1.
  • Double-stranded probes with 'mixed' interstrand arrangements of disclosed monomers are one representative example of this embodiment (e.g., one strand modified with 2'-amino-a- L-LNA monomer 124X, the other strand modified with 2'-amino-a-L-LNA monomer 124Y, Table 13).
  • Significantly negative DA values are observed for these double-stranded probes.
  • probes with 'mixed' +1 zippers display DA values between -19.5 °C and -23.0 °C (Table 13).
  • probes with +1 zippers composed of different monomers display significant dsDNA-targeting potential and enable targeting of dsDNA-regions as described in FIG 1.
  • aAT m change in T m values relative to unmodified reference duplex D1:D2 (T m - 29.5 °C); see Table 8 for experimental conditions.
  • Certain embodiments entail double-stranded probes where one strand is modified with one or more monomers comprising a thymine nucleobase, and the other strand is modified is with one or more monomers comprising an adenine nucleobase.
  • Particular embodiments of a double-stranded probe where one strand is modified with a thymine monomer (126W or 126X) and the other strand is modified is with an adenine monomer (124X or 124Y) are given in (Tables 14-17 ).
  • Probes with 0 or +2 interstrand monomer arrangements generally display significantly negative DA-values (DA-values between -22 °C to -10 °C), indicating significant potential for targeting of double-stranded nucleic acid targets, more commonly dsDNA, via the method shown in FIGS 1-2.
  • Probes with -2 interstrand arrangements display DA-values ⁇ 0 °C.
  • probes with 0, +1 or +2 interstrand zipper arrangements of disclosed monomers may display significant potential for targeting of double-stranded nucleic acids, more commonly dsDNA, and may enable targeting of dsDN A as shown FIG 1.
  • Additional embodiments include double-stranded probes with +1 interstrand zipper arrangements of monomers 208W-Z (Table 18).
  • probe-target duplex involving upper (column 4 of 6) or lower probe strand (column 5 of 6) are given.
  • probes with +1 interstrand zipper arrangement of the monomers (column 3) display similar or lower thermostability than model DNA duplex target (column 2; note negative delta Tm values in column 3), while probe-target duplexes (columns 4 and 5) may be greatly stabilized (note highly positive delta Tm values).
  • probes with +1 interstrand arrangements of monomers 120Y/208X/208Y/208Z display significantly positive thermal advantage (TA) values and, thus, significant potential for targeting of double-stranded nucleic acid targets, more commonly dsDNA, via the method outlined in FIGS 1-2.
  • TA thermal advantage
  • probes with +1 interstrand zipper arrangements of certain disclosed monomers display significantly positive thermal advantage (TA) values and, thus, significant potential for targeting of double-stranded nucleic acid targets, more commonly dsDNA, via the method outlined in FIGS 1-2.
  • TA thermal advantage
  • Table 20 provides thermal denaturation temperatures for a model DNA duplex target (column 6 of 7), probe (column 5 of 7), probe-target duplexes [i.e., model products from dsDNA recognition] involving upper (column 3 of 7) or lower probe strand (column 4 of 7).
  • Probes with one + 1 interstrand zipper arrangement of the monomers display similar or lower thermostability than unmodified dsDNA (representing the target; note negative delta Tm values in column 5), while probe-target duplexes (columns 3 and 4) display far greater stabilization (note highly positive delta Tm values).
  • probes with other interstrand arrangements may display less positive (or even negative) TA values and may therefore display less dsDNA-targeting potential (i.e., entriesin this Table).
  • Probes with two or more +1 interstrand arrangements of the disclosed monomers may display very high TA- values, suggesting significant potential for targeting of double-stranded targets, more commonly, dsDNA target regions, via the method shown in FIGS 1-2.
  • probes with 0-arrangements of disclosed monomers in some particular cases, monomers 120Y and 120"W.
  • Such probes may display variable TA values ranging from -1 °C to +27 °C, demonstrating that said probes may display significant potential for targeting of double-stranded nucleic acid targets, more commonly dsDNA targets, via the method outlined in FIGS 1-2.
  • T 120Y and A is 120'W
  • Tables 22-24 outline thermal denaturation properties of additional working embodiments of yet further probes comprising unlocked monomers targeting different DNA regions, i.e., second insulin [INSB], PPAR gamma and CEBP promotors). Yet again, the following thermal denaturation temperatures are given: model DNA duplex target (column 5 of 6), probe (column 4 of 6), probe- target duplex [product from dsDNA recognition] involving upper (column 2 of 6) or lower probe strand (column 3 of 6). Probes with one or more +1 interstrand zipper arrangement of the monomers display variable thermostability (ranging from strongly destabilized to moderately stabilized relative to unmodified dsDNA (note delta Tm values from -18 to +8 C, column 4).
  • Probe-target duplexes (columns 2 and 3) are greatly stabilized (note highly positive delta Tm values). All probes display positive thermal advantage (TA). Probes with two or more +1 interstrand monomers arrangements display larger TA values (and thus, greater dsDNA-targeting potential) than probes with a single +1 interstrand monomers arrangement. Without being limited to a single theory of operation, probes with more than one +1 zipper arrangement of monomers facilitate dsDNA-targeting.
  • Tables 25-27 describe the thermal denaturation properties of probes that may be used for gender determination of individual cells or multicellular assemblies from certain animals and humans; more commonly somatic cells, sperm cells or embryos from certain animals and humans; even more commonly, somatic cells, sperm cells or embryos from bovine.
  • probes display thermostabilities that range from significantly lower to moderately higher than corresponding unmodified double-stranded DNA targets (note delta Tm values from -13 C to +9; column 4), while probe-target duplexes (column 2 and 3) are significantly more thermostable (range from +5 to +24 C). Accordingly, all of the probes (which have between two to five +1 zipper monomer arrangements) display significantly positive TA-values suggesting significant potential for targeting of double-stranded nucleic acid targets, more commonly dsDNA. Table 25
  • Table 26 shows thermal denaturation properties and TA-values for probes modified with unlocked monomer 120Q. Similar patters as seen for other disclosed monomers are observed, i.e., probes display relatively low thermostability while probe-target duplexes are significantly more thermostable. Probes containing one or more +1 zipper arrangement of unlocked monomer 120Q therefore display significantly positive TA-values and therefore significant potential for targeting of double-stranded nucleic acid targets, more commonly dsDNA targets.
  • Particular embodiments entail double-stranded probes with certain zipper arrangements of monomers comprising so-called pseudo-complementary nucleobases (e.g., such as 2-thiouracil, 2,6- diamonopurines, inosine and pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one), more commonly, +1 zipper arrangements of monomers comprising pseudo-complementary nucleobases, even more commonly, + 1 zipper arrangements of monomers such as 270. Examples of working examples of these particular embodiments are given in Table 27 below.
  • pseudo-complementary nucleobases e.g., such as 2-thiouracil, 2,6- diamonopurines, inosine and pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one
  • +1 zipper arrangements of monomers comprising pseudo-complementary nucleobases even more commonly, + 1 zipper arrangements of monomers such as 270. Examples of working examples of these particular embodiments are
  • nucleotide opposite of the disclosed monomer comprising a pseudo-complementary nucleobase is a nucleotide or disclosed monomer comprising a pseudo-complementary nucleobase (e.g., such as 2-thiouracil, 2,6-diamonopurines, inosine and pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one).
  • a pseudo-complementary nucleobase e.g., such as 2-thiouracil, 2,6-diamonopurines, inosine and pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one.
  • Particular embodiments entail double-stranded probes simultaneously comprising one or more arrangements of disclosed monomers and one or more non-pairing monomers (FIG. 3b ).
  • Working examples of such embodiments are provided below.
  • Table 28 concerns thermal hybridization properties and dsDNA-targeting potential of 18-mer probes containing non-pairing bulges with a general structure:
  • L denotes the non-pairing monomer; in particular working examples, L is selected from monomers 402-4, 402-9 and 402-N.
  • L is selected from monomers 402-4, 402-9 and 402-N.
  • "4", “9” and “N” denotes a single incorporation of 402-4, 402-9 and 402-N, respectively.
  • "444", "999” and “NNN” denotes three consecutive incorporations of 402-4, 402-9 and 402-N, respectively.
  • “Up”, “down” and “both” in the third column denotes whether bulged monomer(s) were included only in the upper probe strand, only in the lower probe strand or in both probe strands, respectively.
  • probes comprising one or two non-pairing bulges display positive TA-values; in the working examples shown below, several of the probes comprising one or two non-pairing bulges display significantly similar or larger TA-values than the control probe shown in entry 1, suggesting significant potential for targeting of double-stranded nucleic acid targets via the method disclosed in FIGS 1-2, more commonly dsDNA.
  • Table 28
  • probes with one or more bulges toward probe termini display significant potential for targeting of double-stranded nucleic acid targets, more commonly dsDNA, via the method shown in FIGS 1-2.
  • the disclosed probe is capable of associating with a target.
  • the disclosed probe comprises at least one pair of monomers that are arranged in a +/-n zipper arrangement, which allows the monomers to affect the thermostability of the probe.
  • Certain zipper arrangements i.e., -1, 0, +1 and +2 zipper arrangements but more commonly +1 zipper arrangements provides the probe with sufficient thermodynamic instability to cause the two strands of the probe duplex to dissociate in the presence of a significantly complementary double-stranded nucleic acid target, more commonly dsDNA, and thereby associate with a target nucleic acid.
  • the two separated strands of the probe will partake in Watson- Crick base pairing with the nucleotides of the target nucleic acid, more commonly the two strands of a dsDNA.
  • the probe may be used in diagnostic techniques, such as identification of biomarkers, oncogenes, gender-specific genes, etc., and/or direct detection of double-stranded DNA in living cells, embryos, organs and tissues and/or induction of site-specific mutagenesis, recombination or repair of genomic DNA and site-specific modulation of gene expression (i.e. up- or down regulation).
  • the probe is not limited to use in these techniques, as this list is meant only to be exemplary and not limiting.
  • the probe may be pre-annealed using methods known to those of ordinary skill in the art. The pre-annealed probe may then be added to a target, such as a double stranded DNA target.
  • the disclosed probe may be designed to have at least one pair of monomers.
  • the probe is designed to have two pairs of monomers, wherein the two pairs may be identical or different, and are separated by zero or more natural or non-natural nucleotides, such as disclosed monomers herein.
  • a pair of monomers comprises at least two unlocked or locked monomers functionalized with an intercalator and arranged in a +1 zipper arrangement.
  • the probe may be designed to target a particular isosequential nucleic acid target - whether synthetic or biological, such as any of those disclosed herein.
  • the nucleic acid target may be the SP6 and T7 promoters on PGEM-Teasy plasmids that may or may not overlap with a transcription start site.
  • the pGEM-T-Easy vector containing insb-cDNA was linearized with either Spel or SacII and used for in vitro transcription reactions to synthesize cRNA driven by T7 or SP6 promoters, respectively (FIG. 18).
  • the linearized plasmids were incubated with dsDNA-targeting agents as follows: either positive control (commercial Zorro LNA), targeting the SP6 promoter, or the disclosed probe selected to target the SP6 or T7 promoter (FIG. 19).
  • positive control commercial Zorro LNA
  • the disclosed probe selected to target the SP6 or T7 promoter (FIG. 19).
  • in vitro transcription was initiated by incubating with ribonucleotide triphosphates, buffer, and T7 or SP6 polymerases.
  • cRNA products were reverse transcribed to cDNA.
  • Primers designed to detect a 240 base insert amplicon were used in end-point PCR and the product was resolved by gel electrophoresis. The results of this particular disclosed embodiment are illustrated in FIG.
  • lanes 1 and 9 illustrate the DNA ladder (100 bp increments); lane 2 illustrates the T7-driven product formed in Spel digested plasmid in the absence of either the control or the probe; lane 3 illustrates the SacII digested plasmid, which does not yield T7 -driven product; lane 4 illustrates that a particular embodiment of the probe binds to the T7 promoter and prevents formation of T7-driven product in Spel digested plasmid; and lane 5 illustrates that the SP6-driven product is formed in SacII digested plasmid in the absence of a either the positive control or the probe. Also referring to FIG.
  • lanes 6-8 illustrate that Zorro LNA (In 6), one embodiment of the disclosed probe (In 7) or a different embodiment of the disclosed probe (In 8) bind to the SP6 promoter and prevent formation of SP6-driven product in SacII treated plasmid.
  • Other targets are contemplated by the disclosed method, such as gene knockdown in live cell lines targeting chromosomal progesterone receptor, estrogen receptors, and any other biologically relevant targets.
  • the disclosed probe may be used for animal sexing, such as sexing of ungulates, ruminates, and more particularly bovines, equines and porcines, as the probe may be designed to selectively targeting gender-specific DNA regions. Examples of gender-specific DNA regions are known in the art. See, for example, WO 2009079456 and Brown, Kim H., Irvin R. Schultz, J. G. Cloud, and James J. Nagler (2008) "Aneuploid Sperm Formation in Rainbow Trout
  • the probe may be designed to comprise one or more pairs of the disclosed monomers and have a sequence of nucleotides that is isosequential with a particular gene of a target cell that is specific for certain genetic traits, such as gender.
  • the probe comprises one or more pairs of disclosed monomers selected from embodiments of monomers disclosed herein, such as those of Scheme 16, and may contain zero or more non-pairing monomers such as 402-4, 402-9 or 402-9.
  • the probe may be used to determine the gender of animals and cells (in particular sperm cells), organs, tissues and embryos thereof. Particular embodiments enable gender determination of unadulterated early-stage embryos from animals used in food production and sport breeding.
  • Beta TC-6 cells (ATCC, CRL-11506) were co-transfected with [pGL4.10 (luc2/-374insb)] and an internal transfection control vector [pGL4.74 (hRluc/TK) (FIG. 21).
  • a probe comprising at least one pair of locked monomers targeting the insb promotor was transfected 24h after plasmid co-transfection.
  • 2 ⁇ g of the probe per well was used (6-well plate format).
  • the cells were harvested 24h after probe addition (90-100% confluency) and assayed for Firefly and Renilla luciferase (enzyme) activity using a dual luciferase assay system (Promega) to determine the efficacy and specificity of probe-mediated antigene activity.
  • Firefly luciferase activity was normalized to Renilla luciferase activity to correct for transfection variation. Normalized Firefly luciferase activity is expressed relative to a scrambled control probe.
  • the probe may be used to target an isosequential double-stranded DNA duplex or structured analogs hereof.
  • two strands of an isosequential target duplex may be connected through a linker, such as a polynucleotide, to produce a duplex having a hairpin configuration (FIG. 24).
  • the linker comprises ten thymidines, with the stem of the hairpin target being the primary region recognized by the probe.
  • the nucleic acid target may or may not comprise one or more polypurine units.
  • the intramolecular nature of the target duplex of the hairpin structure increases the T m value of the duplex, rendering it a more challenging target than linear dsDNA targets.
  • the ability of the probe to recognize and invade (or associate with) the complex may be analyzed using an electrophoretic mobility shift assay.
  • the ability of a (+1) interstrand zipper probe comprising at least one pair of monomers was used to target a hairpin target.
  • hairpin invasion by the disclosed probe resulted in a probe-target complex having a slower migration rate, such as that illustrated in lanes A-D of FIG. 25.
  • the concentration of the probe added to the target affects the ability of the probe-target complex to be formed.
  • an excess of about 5-fold to about 500-fold of the disclosed probe may result in probe- target complex formation; even more typically, an excess of from about 5-fold to about 50-fold of the disclosed probe will result in significant probe-target complex formation.
  • FIG. 1 hairpin invasion by the disclosed probe resulted in a probe-target complex having a slower migration rate, such as that illustrated in lanes A-D of FIG. 25.
  • the concentration of the probe added to the target affects the ability of the probe-target complex to be formed.
  • an excess of about 5-fold to about 500-fold of the disclosed probe may result in probe- target complex formation; even more typically, an excess of
  • lanes A-E represent samples in which a varying excess of the probe is used, with lane A representing a 500-fold excess of the probe, lane B representing a 100-fold excess of the probe, lane C representing a 50-fold excess of the probe, lane D representing a 10-fold excess of the probe, and lane E representing a 5-fold excess of the probe.
  • any of the disclosed monomers may be incorporated into the probe.
  • monomers 124X, 124Y, 126W, 126X, 126Y, 126Z, 120Q, 120S, 120V, 120Y, and 120' W were used to form a (+1) interstrand zipper within the probe. Working examples of these embodiments are given in FIG 30.
  • probes with +1 arrangements of the disclosed monomers recognize a structured and digoxigenin-labeled dsDNA target comprised of an isosequential double-stranded target region, which is linked on one side by a single-stranded Ti 0 loop (SL1, FIG. 30b).
  • SL single-stranded Ti 0 loop
  • the ability of the disclosed probe to invade, or associate with, the target may be evaluated by comparing its reactivity with that of control probes. For example, incubation of the mixed-sequence hairpin target with an unmodified isosequential DNA duplex did not show any complex formation, even with up to a 500-fold excess of the unmodified DNA duplex. In addition, incubation of the hairpin target with the either single-stranded probe precursor that comprises a double-stranded probe did not exhibit substantial complex formation (FIGS. 26 D & E). Furthermore, the sequence specificity of the probe may be determined by methods known to those of ordinary skill in the art. In exemplary embodiments, singly and doubly mutated, but perfectly base paired hairpins were targeted. In these particular embodiments, the probe- target complex was not observed, even at a 100-fold (5uM) excess of the probe, thereby demonstrating high target specificity (FIGS. 26 A & B).
  • Reactions were conducted under argon whenever anhydrous solvents were used. Reactions were monitored by TLC using silica gel coated plates with a fluorescence indicator (SiO 2 -60, F-254) which were visualized a) under UV light and/or b) by dipping in 5% cone. H 2 S0 4 in absolute ethanol (v/v) followed by heating. Silica gel column chromatography was performed with Silica gel 60 (particle size 0.040-0.063 mm) using moderate pressure (pressure ball). Evaporation of solvents was carried out under reduced pressure at temperatures below 45 °C.
  • TMSOTf (43.1 mL, 0.24 mol) was added and the reaction mixture heated at reflux for 70h. After cooling to rt. the mixture was poured into sat. aq. NaHCOs /crushed ice (500 mL, 1 : 1, v/v). To control effervescence additional crushed ice (400 mL) was added, and the mixture was stirred for 30 min during which a precipitate was formed. The precipitate was removed by filtration and the filtrate was washed with CH 2 C1 2 (1.5 L). The combined organic phase was washed with sat. aq. NaHC0 3 (2 x 500 mL) and the aqueous phase back-extracted with CH 2 C1 2 (2 x 500 mL).
  • guanidine/guanidinium nitrate (G/GHNO 3 ) was prepared according to the published procedure by dissolving guanidinium nitrate (4.91 g, 40.2 mmol) in a mixture of
  • Alcohol 34 (18. 6 g, 30 mmol) was co- evaporated with pyridine (50 mL), and subsequently dissolved in an. CH 2 CI 2 (200 mL). The mixture was then cooled to -78 °C, and pyridine (7.3 mL, 90 mmol) and trifluoromethanesulfonyl anhydride (9.90 mL, 60 mmol) was added.
  • Amine derivative 40 (8.68 g, 15.8 mmol) was co-evaporated with pyridine (2 x 20 mL) and dissolved in anhydrous CH 2 CI 2 (200 mL).
  • reaction mixture was stirred at rt. for 21h, whereupon additional 2-cyanoethyl-A ⁇ A ⁇ -(diisopropyl)-phosphoramidochloridite (0.05 mL, 0.06 mmol) was added.
  • the reaction mixture was then stirred for 4h followed by addition of abs. EtOH (2 mL). Subsequently the mixture was diluted with CH 2 CI 2 (10 mL) and the organic phase was washed sequentially with sat. aq. NaHC0 3 (10 mL) and brine (10 mL).
  • the reaction mixture was stirred at rt. for 45h, whereafter it was diluted with CH 2 C1 2 (50 mL) and washed with water (20 mL). The two phases were separated and the aqueous phase back-extracted with CH 2 C1 2 (3 x 50 mL). The combined organic phase was evaporated under reduced pressure and the resulting residue was purified by silica gel column chromatography (0-99% EtOAc and 1 % pyridine in petroleum, v/v). The resulting product was co-evaporated with abs. EtOH:toluene (2 x 50 ml, 1 : 1, v/v) affording target nucleoside 51Y as a yellow solid (343 mg, 64%).
  • the reaction mixture was stirred at rt. for 2.5h, whereafter it was diluted with CH 2 C1 2 (20 mL) and washed with water (2 x 10 mL). The two phases were separated and the aqueous phase back-extracted with CH 2 C1 2 (10 mL). The combined organic phase was evaporated under reduced pressure and the resulting residue was purified by silica gel column chromatography (0-4% MeOH in CH 2 C1 2 , v/v) affording target nucleoside 51Z as a white solid (266 mg, 79%).
  • Synthesis of probes with locked monomers 124W-124Y Syntheses of probes containing incorporations of locked phosphoramidites 52W, 52X, and 52Y were performed on an automated DNA synthesizer (0.2 ⁇ scale) using the following hand coupling conditions (activator; coupling time; approximate coupling yield): Monomer 124W (pyridinium hydrochloride; 30 min; -82%), monomers 124X and 124Y (pyridinium hydrochloride; 15 min; -95%). The probes were deprotected using 32% aq. NH 3 at 55 °C for 2h. Purification of probes (till at least 75% purity) was performed by RP-HPLC (DMT-ON), followed by detritylation (80% aq. AcOH, 20 min) and precipitation (abs.
  • RP-HPLC purification of oligonucleotides was performed using a Waters Prep LC 4000 system equipped with an Xterra MS C18-column (10 ⁇ , 300 mm X 7.8 mm).
  • a representative RP-HPLC gradient protocol for purification of oligonucleotides with DMT-ON is to use an isocratic hold of 100% A-buffer for 5 min followed by a linear gradient to 55% B-buffer over 75 min at a flow rate of 1.0 mL/min (A-buffer: 95% 0.1 M NH 4 HC0 3 , 5% CH 3 CN; B-buffer: 25% 0.1 M NH 4 HC0 3 , 75% CH 3 CN).
  • composition of the probes was verified by MALDI-MS analysis (Table 32) whereas the purity (>80%, unless stated otherwise) was verified by ion-exchange HPLC using a LaChrom L-7000 system (VWR International) equipped with a Gen-Pak Fax column (100 mm X 4.6 mm).
  • a representative protocol involves the use of an isocratic hold of 95% A-buffer for 5 min, followed by a linear gradient to 70% B-buffer over 41 min at a flow rate of 0.75 mL/min (A-buffer: 25 mM Tris-Cl, 1 mM EDTA, pH 8.0; B-buffer: 1 M NaCl).
  • Single-stranded probes that are modified with 124X and 124Y show higher thermal affinity toward single-stranded DNA than toward single-stranded RNA targets (Tables 33 and 34). This DNA selectivity makes these monomers useful as probes for targeting double stranded DNA.

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Abstract

Les modes de réalisation de la présente invention concernent une sonde comprenant une ou plusieurs paires de monomères aptes à viser un acide nucléique cible. Les deux monomères peuvent être agencés de manière à favoriser l'instabilité thermique du complexe sonde, afin de produire une sonde apte à localiser et/ou à détecter une cible. La sonde peut également comprendre un ou plusieurs nucléotides naturels ou non naturels aptes à former des paires de bases de Watson-Crick avec un acide nucléique isoséquentiel cible. Les modes de réalisation particuliers décrits concernent un procédé d'utilisation de la sonde selon l'invention pour le ciblage des acides nucléiques. Dans les modes de réalisation particuliers décrits, la sonde peut être incubée avec un acide nucléique cible et ensuite détectée.
PCT/US2012/047442 2011-07-19 2012-07-19 Modes de réalisation d'une sonde et procédé de ciblage d'acides nucléiques WO2013013068A2 (fr)

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US14/233,375 US9885082B2 (en) 2011-07-19 2012-07-19 Embodiments of a probe and method for targeting nucleic acids
RU2014106024/04A RU2014106024A (ru) 2011-07-19 2012-07-19 Варианты осуществления зонда и способы направленного действия на нуклеиновые кислоты
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MX2014000797A MX2014000797A (es) 2011-07-19 2012-07-19 Sonda y metodo para direccionar acidos nucleicos.
BR112014001075A BR112014001075A2 (pt) 2011-07-19 2012-07-19 sonda, seu uso, método in vitro para associar uma sonda com um alvo e kit
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CN106636454A (zh) * 2015-10-28 2017-05-10 中国科学院上海巴斯德研究所 一种同时检测人冠状病毒229e，oc43，nl63和hku1的实时荧光多重rt-pcr方法
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EP2734525A2 (fr) 2014-05-28
AU2012283994A1 (en) 2014-03-06
US9885082B2 (en) 2018-02-06
CA2842103A1 (fr) 2013-01-24
NZ621347A (en) 2016-03-31
WO2013013068A3 (fr) 2013-03-28
RU2014106024A (ru) 2015-08-27
US20140220573A1 (en) 2014-08-07
CN103958519A (zh) 2014-07-30
EP2734525A4 (fr) 2015-03-04
AR088140A1 (es) 2014-05-14
WO2013013068A4 (fr) 2013-05-30
MX2014000797A (es) 2014-07-09

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